Methods of Use of Seed-Origin Endophyte Populations

ABSTRACT

This application relates to methods and materials for providing a benefit to a seed or seedling of an agricultural plant (e.g., an agricultural grass plant), or the agricultural plant derived from the seed or seedling. For example, this application provides purified bacterial populations that include novel seed-origin bacterial endophytes, and synthetic combinations of seeds and/or seedlings (e.g., cereal seeds and/or seedlings) with heterologous seed-derived bacterial endophytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/704,891, filed May 5, 2015, (allowed), which is a continuation ofU.S. application Ser. No. 14/316,469, filed Jun. 26, 2014, now U.S. Pat.No. 9,113,636, issued Aug. 25, 2015, which claims priority toProvisional Application Ser. No. 61/957,255, filed Jun. 26, 2013;Provisional Application Ser. No. 61/959,859, filed Sep. 4, 2013;Provisional Application Ser. No. 61/959,847, filed Sep. 4, 2013;Provisional Application Ser. No. 61/959,858, filed Sep. 4, 2013;Provisional Application Ser. No. 61/959,854, filed Sep. 4, 2013;Provisional Application Ser. No. 61/959,861, filed Sep. 4, 2013;Provisional Application Ser. No. 61/959,870, filed Sep. 4, 2013; andProvisional Application Ser. No. 61/935,761, filed Feb. 4, 2014, thedisclosures of which are incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 4, 2016, isnamed 33158_US_CRF_sequencelisting.txt, includes 1470 sequences, and is1,951,930 bytes in size.

TECHNICAL FIELD

This application relates to methods and materials for providing abenefit to a seed or seedling of an agricultural plant such as anagricultural grass plant, particularly a cereal, or an agriculturalplant such as an agricultural grass plant derived from the seed orseedling. For example, this application provides purified bacterialpopulations that include novel seed-origin bacterial endophytes, andsynthetic combinations of seeds and/or seedlings with heterologousseed-derived bacterial endophytes. Such seed-origin bacterial endophytescan provide beneficial properties to the seed, seedling, or theagricultural plant derived from the seed or seedling, includingmetabolism, transcription, proteome alterations, morphology, and theresilience to a variety of environmental stresses, and combination ofsuch properties.

BACKGROUND

Economically-, environmentally-, and socially-sustainable approaches toagriculture and food production are required to meet the needs of agrowing global population. By 2050 the United Nations' Food andAgriculture Organization projects that total food production mustincrease by 70% to meet the needs of the growing population, a challengethat is exacerbated by numerous factors, including diminishingfreshwater resources, increasing competition for arable land, risingenergy prices, increasing input costs, and the likely need for crops toadapt to the pressures of a drier, hotter, and more extreme globalclimate. The need to grow nearly twice as much food with less water inmore arid climates is driving a critical need for innovations in cropwater use efficiency and temperature tolerance.

Today, crop performance is optimized primarily via technologies directedtowards the interplay between crop genotype (e.g., plant breeding,genetically-modified (GM) crops) and its surrounding environment (e.g.,fertilizer, synthetic herbicides, pesticides). While these paradigmshave assisted in doubling global food production in the past fiftyyears, yield growth rates have stalled in many major crops, and shiftsin the climate have been linked to production instability and declinesin important crops such as wheat, driving an urgent need for novelsolutions to crop yield improvement. In addition to their longdevelopment and regulatory timelines, public fears of GM-crops andsynthetic chemicals has challenged their use in many key crops andcountries, resulting in a complete lack of acceptance for GM traits inwheat and the exclusion of GM crops and many synthetic chemistries fromEuropean markets. Thus, there is a significant need for innovative,effective, environmentally-sustainable, and publically-acceptableapproaches to improving the yield and resilience of crops to severedrought and heat stresses.

Improvement of crop resilience to heat and drought stress has provenchallenging for conventional genetic and chemical paradigms for cropimprovement. This challenge is in part due to the complex, network-levelchanges that arise during exposure to these stresses. For example,plants under such stress can succumb to a variety of physiological anddevelopmental damages, including dehydration, elevated reactive oxygenspecies, impairment of photosynthetic carbon assimilation, inhibition oftranslocation of assimilates, increased respiration, reduced organ sizedue to a decrease in the duration of developmental phases, disruption ofseed development, and a reduction in fertility.

Like humans, who utilize a complement of beneficial microbial symbionts,plants have been purported to derive a benefit from the vast array ofbacteria and fungi that live both within and around their tissues inorder to support the plant's health and growth. As described in detailherein, endophytes are fungal or bacterial organisms that live withinplants. Bacterial endophytes, such as Firmicutes, Actinobacteria,Proteobacteria, Bacteroidetes, and Verrucomicrobia, appear to inhabitvarious host plant tissues and have been isolated from plant leaves,stems, and roots.

To date, a small number of these symbiotic endophyte-host relationshipshave been analyzed in limited studies to provide fitness benefits tomodel host plants within controlled laboratory settings, such asenhancement of biomass production (i.e., yield) and nutrition, increasedtolerance to stress such as drought and pests. Yet, such endophytes havebeen demonstrated to be ineffective or of limited efficacy in conferringbenefits to a variety of agriculturally-important plants such as moderncereals; as such, they do not adequately address the need to provideimproved yield and tolerance to environmental stresses present in manyagricultural situations for such crops, particularly drought and heat.

Thus, there is a need for compositions and methods of providing cerealcrops with improved yield and resistance to various environmentalstresses. Provided herein are novel compositions of symbionts, bacterialand fungal endophytes, as well as novel symbiont-plant compositions,created based on the analysis of the key properties that enhance theutility and commercialization of an endophytic composition.

SUMMARY

The present invention is based, in part, on the surprising discoverythat endophytic microbes can be found in dry mature seeds of plants. Theinventors have isolated and extensively characterized a large number ofbacterial and fungal endophytes of seed-origin that are able to colonizeagricultural grass plants and to provide beneficial traits to theseplants. As such, provided herein are purified bacterial and fungalpopulations that contain one or more populations of seed-originendophytes, particularly bacterial endophytes (herein referred to asseed-original bacterial endophytes), compositions (e.g., agriculturalformulations and articles of manufacture) that include such purifiedbacterial populations, as well as synthetic combinations of suchpurified bacterial populations in association with seeds or seedlings ofan agricultural cereal plant and other agricultural products, includingseeds. In addition, provided herein are methods of using suchseed-origin bacterial endophytes to prepare synthetic combinations,agricultural formulations, articles of manufacture, or otheragricultural products, and to provide benefits to agricultural cerealplants. Seed-derived endophytes can confer significant advantages tocereal crops, spanning growth under normal and stressed conditions,altered expression of key plant hormones, altered expression of keytranscripts in the plant, and other desirable features.

As described herein, beneficial microbes can be robustly derived fromagricultural seeds, cultured, administered heterologously toagricultural cereal seeds or seedlings, and colonize the resulting planttissues with high efficiency to confer multiple beneficial properties,that are durably retained in the plants and their progeny. This issurprising given the historical observed variability in microbeisolation from healthy seeds and the previous observations ofinefficient seed pathogen colonization of a plant host's tissues.Further, the ability of heterologously disposed seed-origin bacterialendophytes to colonize seeds and seedlings from the exterior surface ofseeds is surprising given that such endophytes can be isolated fromwithin internal seed tissues and therefore may not natively need thecapacity to penetrate and invade into internal host tissues in theirnatural state.

Seed-origin bacterial endophytes are heterologously disposed ontoseedlings of a distinct cultivar, species, or cereal crop type andconfer benefits to those new recipients. For example, seed-originbacterial endophytes from corn cultivars are heterologously provided towheat cultivars to confer a benefit. This is surprising given the priorobservations of distinct microbiome preferences in distinct plant andmammalian hosts and, in particular, the likelihood that microbes derivedfrom seeds may have been co-evolved to be specialized to a particularhost.

In one aspect, the invention features a method for treating seeds. Themethod includes contacting the surface of a plurality of Gramineaeagricultural plant seeds with a formulation comprising a purifiedbacterial population at a concentration of at least about 10² CFU/ml ina liquid formulation or about 10² CFU/gm in a non-liquid formulation,where at least 10% of the CFUs present in the formulation comprise apreferred seed-origin bacterial endophyte, which exhibits: production ofan auxin, nitrogen fixation, production of an antimicrobial, productionof a siderophore, mineral phosphate solubilization, production of acellulase, production of a chitinase, production of a xylanase, orproduction of acetoin, wherein the seed-origin bacterial endophyte ispresent in the formulation in an amount capable of providing a benefitto the plant seeds or to agricultural plants derived from the plantseeds; and packaging the contacted seeds in a container. The method canfurther include drying the contacted seed. The contacting can includespraying, immersing, coating, encapsulating, or dusting the seeds orseedlings with the formulation.

The invention also features a method for treating seedlings. The methodincludes contacting foliage or the rhizosphere of a plurality ofGramineae agricultural plant seedlings with a formulation comprising apurified bacterial population at a concentration of at least about 10²CFU/ml in a liquid formulation or about 10² CFU/g in a non-liquidformulation, wherein at least 10% of the CFUs present in the formulationcomprise a seed-origin bacterial endophyte exhibiting production of anauxin, nitrogen fixation, production of an antimicrobial compound,production of a siderophore, mineral phosphate solubilization,production of a cellulase, production of a chitinase, production of axylanase, or production of acetoin, and wherein the seed-originbacterial endophyte is present in the formulation in an amount capableof providing a benefit to the seedlings or to agricultural plantsderived from the seedlings; and growing the contacted seedlings. Thecontacting can include spraying, immersing, coating, encapsulating, ordusting the seeds or seedlings with the formulation.

In another aspect, a method for modulating a Gramineae plant trait isfeatured. The method includes applying to vegetation (e.g., corn, wheat,rice, or barley seedlings) or an area adjacent the vegetation, aformulation that includes a purified bacterial population at aconcentration of at least about 10² CFU/ml in a liquid formulation orabout 10² CFU/g in a non-liquid formulation, at least 10% of the CFUspresent in the formulation comprising a seed-origin bacterial endophyteexhibiting: production of an auxin, nitrogen fixation, production of anantimicrobial compound, production of a siderophore, mineral phosphatesolubilization, production of a cellulase, production of a chitinase,production of a xylanase, or production of acetoin, and combinations oftwo or more thereof, wherein the formulation is capable of providing abenefit to the vegetation, or to a crop produced from the vegetation.

A method for modulating a Gramineae plant trait is featured thatincludes applying a formulation to soil, the formulation comprising apurified bacterial population at a concentration of at least about 10²CFU/g, at least 10% of the CFUs present in the formulation comprising aseed-origin bacterial endophyte exhibiting: production of an auxin,nitrogen fixation, production of an antimicrobial compound, productionof a siderophore, mineral phosphate solubilization, production of acellulase, production of a chitinase, production of a xylanase, orproduction of acetoin, and combinations of two or more thereof, whereinthe formulation is capable of providing a benefit to seeds plantedwithin the soil, or to a crop produced from plants grown in the soil.

A method of making an article of manufacture also is featured. Themethod includes applying an agricultural formulation to Gramineae plantseeds, the formulation including a purified bacterial population and anagriculturally acceptable carrier, the bacterial population consistingessentially of a seed-origin bacterial endophyte that exhibits:production of an auxin, nitrogen fixation, production of anantimicrobial, production of a siderophore, mineral phosphatesolubilization, production of a cellulase, production of a chitinase,production of a xylanase, or production of acetoin, or combinations oftwo or more thereof; and packaging the coated seeds in packagingmaterial.

The invention also features a method of identifying a modulator of aplant trait. The method includes applying a bacterial population toseeds of an agricultural plant, the population comprising bacteria ofone or more species of seed-origin bacterial endophytes; measuring atrait in seedlings or plants derived from the seeds, the trait selectedfrom the group consisting of root biomass, root length, height, shootlength, leaf number, water use efficiency, overall biomass, grain yield,photosynthesis rate, tolerance to drought, heat tolerance, salttolerance, resistance to nematode stress, resistance to a fungalpathogen, resistance to a bacterial pathogen, resistance to a viralpathogen, the level of a metabolite, and proteome expression; andidentifying at least one of the traits for which the bacterialpopulation results in a modulation of the trait, relative to referenceseedlings or plants.

In another aspect, a method of identifying a modulator of a plant traitis featured. The method includes applying a bacterial population toseedlings of an agricultural plant, the population comprising bacteriaof one or more species of seed-origin bacterial endophytes; measuring atrait in the seedlings or in plants derived from the seedlings, thetrait selected from the group consisting of root biomass, root length,height, shoot length, leaf number, water use efficiency, overallbiomass, grain yield, photosynthesis rate, tolerance to drought, heattolerance, salt tolerance, resistance to nematode stress, resistance toa fungal pathogen, resistance to a bacterial pathogen, resistance to aviral pathogen; the level of a metabolite, and proteome expression; andidentifying at least one of the traits for which the bacterialpopulation results in a modulation of the trait, relative to referenceseedlings or plants. The modulation can be an increase in root biomass,an increase in root length, an increase in height, an increase in shootlength, an increase in leaf number, an increase in water use efficiency,an increase in overall biomass, an increase in grain yield, an increasein photosynthesis rate, an increase in tolerance to drought, an increasein heat tolerance, an increase in salt tolerance, an increase inresistance to nematode stress, an increase in resistance to a fungalpathogen, an increase in resistance to a bacterial pathogen, an increasein resistance to a viral pathogen, a detectable modulation in the levelof a metabolite, or a detectable modulation in the proteome.

This invention also features a method for treating a cereal seed orseedling. The method includes contacting the exterior surface of acereal seed or seedling with a formulation comprising a purifiedbacterial population, the purified bacterial population comprising at alevel of at least 10% of the CFUs present in the formulation aseed-origin bacterial endophyte capable of at least one of: productionof an auxin, nitrogen fixation, production of an antimicrobial,production of a siderophore, mineral phosphate solubilization,production of a cellulase, production of a chitinase, production of axylanase, or acetoin production, or a combination of two or more, underconditions such that the formulation becomes disposed upon an exteriorsurface of the cereal seed or seedling in a manner effective for theseed-origin bacterial endophyte to provide a benefit to the cereal seedor seedling or to a cereal agricultural plant derived from the seed orseedling, and wherein the seed-origin bacterial endophyte is capable ofhost colonization and/or replication within a tissue of the cerealagricultural plant; and packaging the contacted cereal seed or seedlingin a container. In embodiments in which the cereal seed is contacted,the method further can include drying the contacted cereal seed. Inembodiments in which the cereal seed is contacted, the seed-originbacterial endophyte can be present at a concentration of at least 1CFU/seed on the surface of the contacted cereal seed. Contacting caninclude spraying, immersing, coating, dusting, or dipping the cerealseed or seedling with the formulation. The seed-origin bacterialendophyte can be obtained or obtainable from an interior seedcompartment, e.g., the seed-origin bacterial endophyte can be obtainedor obtainable from an interior seed compartment of a heterologous seedor seedling to the contacted cereal seed or seedling, or can be obtainedor obtainable from an exterior surface of a heterologous seed orseedling to the contacted cereal seed or seedling. The seed-originbacterial endophyte can be heterologous to the microbial populationwithin the contacted cereal seed or seedling. The seed-origin bacterialendophyte can be obtained or obtainable from the interior seedcompartment of a different cultivar, variety or crop as compared to theseed or seedling. The seed-origin bacterial endophyte can be obtained orobtainable from an exterior surface of a different cultivar, variety orcrop as compared to the seed or seedling. The benefit can be heritableby progeny of the agricultural cereal plant derived from the contactedcereal seed or seedling. The seed-origin bacterial endophyte can includea 16S nucleic acid sequence at least 97% identical to a 16S nucleic acidsequence of a bacterial endophyte set forth in Table 1. The seed-originbacterial endophyte can be obtained or obtainable from the seed of arice, maize, wheat, or barley plant. The seed-origin bacterial endophytecan be capable of at least two of: production of an auxin, nitrogenfixation, production of an antimicrobial, production of a siderophore,mineral phosphate solubilization, production of a cellulase, productionof a chitinase, production of a xylanase, and acetoin production.

A method for improving a plant trait in a cereal agricultural plantgrown in a soil region also is featured. The method includes contactingat least a portion of the soil region with a formulation comprising apurified bacterial population, the purified bacterial populationcomprising at a level of at least 10% of the CFUs present in theformulation a seed-origin bacterial endophyte capable of at least oneof: production of an auxin, nitrogen fixation, production of anantimicrobial, production of a siderophore, mineral phosphatesolubilization, production of a cellulase, production of a chitinase,production of a xylanase, and acetoin production, or a combination oftwo or more, under conditions such that the seed-origin bacterialendophyte is capable of providing a benefit to a cereal seed or seedlingplanted within the soil region, or to an agricultural cereal plantderived from the cereal seed or seedling. The method can includeplanting a cereal seed or seedling in the soil region. The seed-originbacterial endophyte can be obtained or obtainable from an interior seedcompartment, e.g., the seed-origin bacterial endophyte can be obtainedor obtainable from an interior seed compartment of a heterologous seedor seedling to the contacted cereal seed or seedling, or can be obtainedor obtainable from an exterior surface of a heterologous seed orseedling to the contacted cereal seed or seedling. The seed-originbacterial endophyte can be exogenous to the microbial population withinthe contacted cereal seed or seedling.

The invention also features a method for planting a field region with anagricultural cereal crop. The method includes obtaining a containercomprising at least 10 synthetic combinations, wherein each syntheticcombination comprises a purified bacterial population in associationwith a cereal seed or seedling, wherein the purified bacterialpopulation comprises a seed-origin bacterial endophyte capable of atleast one of: production of an auxin, nitrogen fixation, production ofan antimicrobial, production of a siderophore, mineral phosphatesolubilization, production of a cellulase, production of a chitinase,production of a xylanase, or acetoin production, or combinations of twoor more thereof, and wherein the seed-origin bacterial endophyte ispresent in an amount effective to provide a benefit to the cereal seedor seedling or the agricultural cereal plant derived from the cerealseed or seedling; and distributing the synthetic combinations from thecontainer in the field region. In any of the methods, the seed-originbacterial endophyte can be obtained or obtainable from an interior seedcompartment, e.g., the seed-origin bacterial endophyte can be obtainedor obtainable from an interior seed compartment of a heterologous seedor seedling to the contacted cereal seed or seedling, or can be obtainedor obtainable from an exterior surface of a heterologous seed orseedling to the contacted cereal seed or seedling. The seed-originbacterial endophyte can be exogenous to the microbial population withinthe contacted cereal seed or seedling.

In any of the methods, the seed-origin bacterial endophyte can bepresent at a concentration of at least 10² CFU/seed on the surface ofthe seeds after contacting.

In any of the methods, the seed-origin bacterial endophyte can beobtained from an interior seed compartment (e.g., cotyledon, plumule,embryo, or endosperm).

In any of the methods, the seed-origin bacterial endophyte can beobtained from a plant species other than the seeds with which theformulation is contacted.

In any of the methods, the seed-origin bacterial endophyte can beobtained from a plant cultivar different from the cultivar of the seedswith which the formulation is contacted.

In any of the methods, the seed-origin bacterial endophyte can beobtained from a surface sterilized seed.

In any of the methods, the benefit can be maternally inherited byprogeny of the contacted plant seeds.

In any of the methods, the seed-origin bacterial endophyte can include a16S nucleic acid sequence having at least 97% sequence identity to a 16Snucleic acid sequence of a bacterial endophyte selected from a genusprovided in Table 1 or a family provided in Table 2.

In any of the methods, the seed-origin bacterial endophyte can include a16S nucleic acid sequence that is less than 97% identical to any 16Snucleic acid sequence shown in Table 1.

In any of the methods, the bacterial population can include a firstseed-origin bacterial endophyte having a first 16S nucleic acid sequenceand a second seed-origin bacterial endophyte having a second 16S nucleicacid sequence, wherein the first and the second 16S nucleic acidsequences are less than 97% identical.

In any of the methods, the bacterial population can include two or morefamilies of seed-origin bacterial endophytes.

In any of the methods, the bacterial population can include two or morespecies of seed-origin bacterial endophytes.

In any of the methods, the seed-origin bacterial endophyte can be anon-Bacillus species and/or a non-Pseudomonas species.

In any of the methods, the seed-origin bacterial endophyte can beobtained from a rice, maize, wheat, or barley seed.

In any of the methods, the seed-origin bacterial endophyte can exhibitat least two of: production of an auxin, nitrogen fixation, productionof an antimicrobial, production of a siderophore, mineral phosphatesolubilization, production of a cellulase, production of a chitinase,production of a xylanase, or production of acetoin, or combinationsthereof.

In any of the methods, the benefit can be selected from the groupconsisting of: increased root biomass, increased root length, increasedheight, increased shoot length, increased leaf number, increased wateruse efficiency, increased overall biomass, increase grain yield,increased photosynthesis rate, increased tolerance to drought, increasedheat tolerance, increased salt tolerance, increased resistance tonematode stress, increased resistance to a fungal pathogen, increasedresistance to a bacterial pathogen, increased resistance to a viralpathogen, a detectable modulation in the level of a metabolite, and adetectable modulation in the proteome, relative to reference seeds oragricultural plants derived from reference seeds. The benefit caninclude a combination of at least two of such benefits.

In another aspect, the invention features a synthetic combination thatincludes a purified bacterial population in association with a pluralityof seeds or seedlings of a Gramineae agricultural plant, wherein thepurified bacterial population comprises a seed-origin bacterialendophyte capable of at least one of: production of an auxin, nitrogenfixation, production of an antimicrobial, production of a siderophore,mineral phosphate solubilization, production of a cellulase, productionof a chitinase, production of a xylanase, or production of acetoin, or acombination of two or more thereof, and wherein the seed-originbacterial endophyte is present in the synthetic combination in an amounteffective to provide a benefit to the seeds or seedlings or the plantsderived from the seeds or seedlings. For example, the effective amountcan be 1×10³ CFU/per seed or from about 1×10² CFU/seed to about 1×10⁸CFU/seed. The benefit can be heritable by progeny of plants derived fromthe seeds or seedlings. The benefit can be selected from the groupconsisting of increased root biomass, increased root length, increasedheight, increased shoot length, increased leaf number, increased wateruse efficiency, increased overall biomass, increase grain yield,increased photosynthesis rate, increased tolerance to drought, increasedheat tolerance, increased salt tolerance, increased resistance tonematode stress, increased resistance to a fungal pathogen, increasedresistance to a bacterial pathogen, increased resistance to a viralpathogen, a detectable modulation in the level of a metabolite, and adetectable modulation in the proteome relative to a reference plant, andcombinations of two or more thereof. The synthetic combination furthercan include one or more additional seed-origin bacterial endophytespecies.

The synthetic combination can include seeds and the seed-originbacterial endophyte can be associated with the seeds as a coating on thesurface of the seeds (e.g., a substantially uniform coating on theseeds). The synthetic combination can include seedlings and theseed-origin bacterial endophyte can be contacted with the seedlings as aspray applied to one or more leaves and/or one or more roots of theseedlings.

The synthetic combination can be disposed within a packaging materialselected from a bag, box, bin, envelope, carton, or container. Thesynthetic combination can include 1000 seed weight amount of seeds,wherein the packaging material optionally comprises a desiccant, andwherein the synthetic combination optionally comprises an anti-fungalagent. The purified bacterial population can be localized on the surfaceof the seeds or seedlings. The seed-origin bacterial endophyte can beobtained from an interior seed compartment.

In another aspect, the invention features an agricultural product thatincludes a 1000 seed weight amount of a synthetic combination producedby the step of contacting a plurality of Gramineae agricultural plantseeds with a liquid formulation comprising a bacterial population at aconcentration of at least 1 CFU per agricultural plant seed, wherein atleast 10% of the CFUs present in the formulation are one or moreseed-origin bacterial endophytes, under conditions such that theformulation is associated with the surface of the seeds in a mannereffective for the seed-origin bacterial endophytes to confer a benefitto the seeds or to a crop comprising a plurality of agricultural plantsproduced from the seeds. The seed-origin bacterial endophytes can bepresent in a concentration of from about 10² to about 10⁵ CFU/ml or fromabout 10⁵ to about 10⁸ CFU/seed. The formulation can be a liquid and thebacterial concentration can be from about 10³ to about 10¹¹ CFU/ml. Theformulation can be a gel or powder and the bacterial concentration canbe from about 10³ to about 10¹¹ CFU/gm.

The invention also features an agricultural formulation that includes apurified bacterial population and an agriculturally acceptable carrier,the bacterial population consisting essentially of a seed-originbacterial endophyte that exhibits: production of an auxin, nitrogenfixation, production of an antimicrobial, production of a siderophore,mineral phosphate solubilization, production of a cellulase, productionof a chitinase, production of a xylanase, or production of acetoin, orcombinations of two or more thereof, where the seed-origin bacterialendophyte present in an amount effective to confer a benefit to aGramineae agricultural plant seed to which the formulation is applied orto an agricultural plant seedling to which the formulation is applied.The seed-origin bacterial endophyte can be obtained from a surfacesterilized seed, from the surface of a seedling, or an unsterilizedseed.

In yet another aspect, the invention features an article of manufacturethat includes packaging material; Gramineae plant seeds within thepackaging material, and at least one species of seed-origin bacterialendophyte associated with the seeds. The article can include two or morespecies of seed-origin bacterial endophytes.

A synthetic combination also is featured that includes a purifiedbacterial population in association with a seed or seedling of a cerealagricultural plant, wherein the purified bacterial population comprisesa seed-origin bacterial endophyte capable of at least one of: productionof an auxin, nitrogen fixation, production of an antimicrobial,production of a siderophore, mineral phosphate solubilization,production of a cellulase, production of a chitinase, production of axylanase, and acetoin production, or a combination of two or morethereof, wherein the seed-origin bacterial endophyte is present in thesynthetic combination in an amount effective to provide a benefit to theseed or seedling or the cereal agricultural plant derived from the seedor seedling. The synthetic combination can be disposed within a packageand is shelf stable. The purified bacterial population can be localizedon the surface of the seed or seedling. The seed-origin bacterialendophyte can be present at a concentration of at least 1 CFU/seed onthe surface of a seed. The seed-origin bacterial endophyte can beobtained or can be obtainable from an interior seed compartment. Theseed-origin bacterial endophyte can be obtained or can be obtainablefrom an interior seed compartment of a heterologous seed or seedling.The seed-origin bacterial endophyte can be obtained or can be obtainablefrom an exterior surface of a heterologous seed or seedling. Theseed-origin bacterial endophyte can be exogenous to the microbialpopulation within the seed or seedling. The benefit can be heritable byprogeny of the agricultural plant. The benefit can include at least twobenefits, wherein the synthetic combination comprises two or moreseed-origin bacterial endophyte.

In another aspect, the invention features a synthetic combination of apurified bacterial population in association with a seed or seedling ofa cereal agricultural plant, wherein the synthetic combination isproduced by the step of contacting the seed or seedling with aformulation comprising a purified bacterial population, wherein thepurified bacterial population comprises an effective amount of aseed-origin bacterial endophyte capable of conferring a benefit on thecontacted seed or seedling or cereal agricultural plant derived from theseed or seedling, the benefit selected from the group consisting of:increased root biomass, increased root length, increased height,increased shoot length, increased leaf number, increased water useefficiency, increased overall biomass, increase grain yield, increasedphotosynthesis rate, increased tolerance to drought, increased heattolerance, increased salt tolerance, increased resistance to nematodestress, increased resistance to a fungal pathogen, increased resistanceto a bacterial pathogen, increased resistance to a viral pathogen, adetectable modulation in the level of a metabolite, and a detectablemodulation in the proteome relative to a reference plant, underconditions such that the formulation becomes disposed upon an exteriorsurface of the seed or seedling in a manner effective for theseed-origin bacterial endophyte to provide the benefit to the seed orseedling or to the cereal agricultural plant derived from the seed orseedling, and wherein the seed-origin bacterial endophyte is capable ofhost colonization and/or replication within a tissue of the cerealagricultural plant.

In yet another aspect, the invention features an agriculturalformulation comprising a purified bacterial population consistingessentially of a seed-origin bacterial endophyte capable of conferringon a seed, seedling, or agricultural plant a benefit selected from:increased tolerance to drought, increased heat tolerance, and increasedsalt tolerance, wherein the seed-origin bacterial endophyte is presentin an amount effective to provide the benefit to a seed or seedling towhich the formulation is administered or to an agricultural plantderived from the seed or seedling to which the formulation isadministered, and an agriculturally acceptable carrier. The seed-originbacterial endophyte is obtained or obtainable from an interior seedcompartment. The seed-origin bacterial endophyte can be obtained orobtainable from an exterior surface of a seed. The seed-origin bacterialendophyte can be heterologous to the microbial population within thecontacted cereal seed or seedling. The seed-origin bacterial endophytecan be obtained or obtainable from the interior seed compartment of adifferent cultivar, variety or crop as compared to the seed or seedling.The seed-origin bacterial endophyte can be obtained or obtainable froman exterior surface of a different cultivar, variety or crop as comparedto the seed or seedling. The seed-origin bacterial endophyte is capableof: production of an auxin, nitrogen fixation, production of anantimicrobial, production of a siderophore, mineral phosphatesolubilization, production of a cellulase, production of a chitinase,production of a xylanase, or acetoin production, or a combination of twoor more thereof. The seed-origin bacterial endophyte can be capable ofgenerating a bacterial network in the agricultural plant derived fromthe seed or seedling or in the seed or seedling to which the formulationis administered, or in the surrounding environment of the plant, seed,or seedling, and wherein the bacterial network is capable of causing adetectable modulation in the level of a metabolite in the seed,seedling, or plant or a detectable modulation in the proteome of theagricultural plant derived from the seed or seedling. The purifiedbacterial population can consist essentially of two seed-originbacterial endophytes.

In any of the methods, synthetic combinations, agricultural products,agricultural formulations, or articles of manufacture, the purifiedbacterial population can consist essentially of two or more species ofseed-origin bacterial endophytes. The purified bacterial population canconsist essentially of seed-origin bacterial endophytes having a 16Snucleic acid sequence at least 97% identical to a bacterial endophyteselected from a genus shown in Table 1 or from a family shown in Table2. The purified bacterial population can consist essentially of asynergistic combination of two seed-origin bacterial endophytes. Thebacterial population can be shelf-stable. The benefit can be selectedfrom the group consisting of: increased root biomass, increased rootlength, increased height, increased shoot length, increased leaf number,increased water use efficiency, increased overall biomass, increasegrain yield, increased photosynthesis rate, increased tolerance todrought, increased heat tolerance, increased salt tolerance, increasedresistance to nematode stress, increased resistance to a fungalpathogen, increased resistance to a bacterial pathogen, increasedresistance to a viral pathogen, a detectable modulation in the level ofa metabolite, and a detectable modulation in the proteome relative to areference plant, or a combination of two or more thereof.

In any of the methods, synthetic combinations, agricultural products,agricultural formulations, or articles of manufacture, the seed-originbacterial endophyte can be a non-spore forming bacterial species. Theseed-origin bacterial endophyte can exhibit: production of auxin,production of an antimicrobial, production of a siderophore, productionof a cellulase, production of a chitinase, production of a xylanase, orproduction of acetoin, or combinations thereof. The seed-originbacterial endophyte can exhibit: production of auxin, production of asiderophore, mineral phosphate solubilization, production of acellulose, production of a chitinase, production of a xylanase, orproduction of acetoin, but does not increase nitrogen fixation relativeto a reference plant. The seed-origin bacterial endophyte can beshelf-stable. The seed-origin bacterial endophyte can be a non-Bacillusspecies and/or a non-Pseudomonas species.

In any of the methods, synthetic combinations, agricultural products,agricultural formulations, or articles of manufacture, the seed-originbacterial endophyte can be obtained from a plant species other than theseeds or seedlings of the synthetic combination. The seed-originbacterial endophyte can be obtained from a plant cultivar different fromthe cultivar of the seeds or seedlings of the synthetic combination. Theseed-origin bacterial endophyte can be obtained from a plant cultivarthat is the same as the cultivar of the seeds or seedlings of thesynthetic combination. The seed-origin bacterial endophyte can beobtained from an exterior surface of a heterologous seed or seedling.

In any of the methods, synthetic combinations, agricultural products,agricultural formulations, or articles of manufacture, the bacterialpopulation can include a seed-origin bacterial endophyte having a 16Snucleic acid sequence that is less than 97% identical to any 16S nucleicacid sequence shown in Table 1. The bacterial population can include aseed-origin bacterial endophyte having a 16S nucleic acid sequence thatis at least 97% identical to a 16S nucleic acid sequence shown inTable 1. The bacterial population can include two or more families ofseed-origin bacterial endophytes. The bacterial population can include afirst seed-origin bacterial endophyte having a first 16S nucleic acidsequence and a second seed-origin bacterial endophyte having a second16S nucleic acid sequence, wherein the first and the second 16S nucleicacid sequences are less than 97% identical.

In any of the methods, synthetic combinations, agricultural products,agricultural formulations, or articles of manufacture, the bacterialpopulation can include a first seed-origin bacterial endophyte and asecond seed-origin bacterial endophyte, wherein the first and secondseed-origin bacterial endophytes are independently capable of at leastone of production of an auxin, nitrogen fixation, production of anantimicrobial, production of a siderophore, mineral phosphatesolubilization, production of a cellulase, production of a chitinase,production of a xylanase, or production of acetoin, or a combination oftwo or more thereof.

In any of the methods, synthetic combinations, agricultural products,agricultural formulations, or articles of manufacture, the bacterialpopulation can include a first seed-origin bacterial endophyte and asecond seed-origin bacterial endophyte, wherein the first and secondseed-origin bacterial endophytes are capable of synergisticallyincreasing at least one of: production of an auxin, nitrogen fixation,production of an antimicrobial, production of a siderophore, mineralphosphate solubilization, production of a cellulase, production of achitinase, production of a xylanase, or production of acetoin, or acombination of two or more thereof, in an amount effective to increasetolerance to drought relative to a reference plant.

In any of the methods, synthetic combinations, agricultural products,agricultural formulations, or articles of manufacture, the bacterialpopulation can include a first seed-origin bacterial endophyte and asecond seed-origin bacterial endophyte, wherein the first and secondseed-origin bacterial endophytes are obtained from the same cultivar.The bacterial population can include a first seed-origin bacterialendophyte and a second seed-origin bacterial endophyte, wherein thefirst and second seed-origin bacterial endophytes are obtained fromdifferent cultivars of the same agricultural plant.

In any of the methods, synthetic combinations, agricultural products,agricultural formulations, or articles of manufacture, the bacterialpopulation can include a first seed-origin bacterial endophyte and asecond seed-origin bacterial endophyte, wherein the first seed-originbacterial endophyte is capable of colonizing a first agricultural planttissue and wherein the second seed-origin bacterial endophyte is capableof colonizing a second agricultural plant tissue not identical to thefirst agricultural plant tissue.

In any of the methods, synthetic combinations, agricultural products,agricultural formulations, or articles of manufacture, the seed-originbacterial endophyte can be obtained or can be obtainable from a barley,rice, maize, or wheat seed. For example, the seed-origin bacterialendophyte can be obtained or can be obtainable from an interiorcompartment of a corn, wheat, or barley seed. The seed-origin bacterialendophyte can be a non-spore forming bacterial species. The seed-originbacterial endophyte can be a non-Bacillus species and/or anon-Pseudomonas species.

In any of the methods, the synthetic combinations, agriculturalproducts, agricultural formulations, or articles of manufacture, theseed-origin bacterial endophyte can exhibit production of auxin,production of an antimicrobial, production of a siderophore, productionof a cellulase, production of a chitinase, production of a xylanase, orproduction of acetoin, or combinations of two or more thereof. Theseed-origin bacterial endophyte can be shelf-stable.

In any of the methods, synthetic combinations, agricultural products,agricultural formulations, or articles of manufacture, the seed-originbacterial endophyte can exhibit production of auxin, production of asiderophore, mineral phosphate solubilization, production of acellulase, production of a chitinase, production of a xylanase, orproduction of acetoin, or combinations of two or more thereof, but doesnot increase nitrogen fixation relative to a reference plant.

In any of the methods, synthetic combinations, agricultural products,agricultural formulations, or articles of manufacture, the bacterialpopulation can include two or more families of seed-origin bacterialendophytes or two or more seed-origin bacterial endophyte species.

In any of the methods, synthetic combinations, agricultural products,agricultural formulations, or articles of manufacture, the seed-originbacterial endophyte can be a non-Bacillus species and/or anon-Pseudomonas species.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. The word “comprising” inthe claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1A is a graph of seed-origin microbes SYM00254 and SYM00284 werecoated on the outside of surface sterilized corn seeds, planted inaxenic conditions and incubated for 7 days to germinate. The dosedelivered to the seed surface was quantified by serial dilution andplating of liquid inoculum, while the microbial population colonizingroots after 7 days of incubation was quantified by macerating roots,serial dilution, plating and colony counting to obtain CFUs per root.

FIG. 1B depicts an alternative approach to observe plant colonization byseed-origin endophytes by tagging the microbes with a kanamycinresistance and GFP containing plasmid. These microbes were coated ontounsterilized maize seed, which was dried in a 50 mL conical tube andstored at room temperature for a week before being planted in cupscontaining sterile sand in a greenhouse. After a week of growth, shootsand roots were macerated using bead beating, serially diluted to 10× and1,000× before plating and colony counting under UV to determine greenfluorescing CFUs per plant on TSA plates containing kanamycin. Controlplant extracts were plated on kanamycin free agar and developed non-GFPcontaining colonies of several un-described microbes.

FIG. 2 contains representative photographs of seedlings. The seedlingsinoculated with SYM-00052 (right) outperformed un-inoculated controlseedlings (left) under salt stress conditions with 100 mM NaCl in media.This provides an example of seed-origin microbes conferring growthpromotion to wheat seeds grown under salt stress.

FIG. 3 contains representative photographs of seedlings. Improved vigoror growth of wheat (above) and corn (below) plants inoculated withseed-borne endophytes was observed. Top left: wheat seeds wereinoculated with SYM00033 and germinated under normal conditions. Topright: wheat seedlings inoculated with SYM00107 show enhanced growthunder drought stress compared to uninoculated controls. Bottom left:SYM00090 inoculated corn seeds show improved growth under heat stresswhen compared with controls. Bottom right: corn seedlings inoculatedwith SYM00596 display enhanced growth under salt stress.

FIG. 4 contains representative photographs depicting seeds of wheat(Briggs cultivar) that were inoculated with the endophyte SYM00057B andgrown under normal conditions (left), grown in the presence of 100 mMNaCl (top right), or under heat stress (bottom right). Increase in rootlength of wheat plants inoculated with seed-borne endophytes.

FIG. 5 contains representative photographs depicting wheat seedsinoculated with a combination of SYM00057B and SYM00016B (bottom row)show enhanced growth under salt stress conditions when compared withcontrols (top row). Combinations of seed-origin microbes confer improvedvigor to wheat.

FIG. 6 contains representative photographs of roots of plants thatgerminated from uninoculated (control) and inoculated seeds (Sym00090)and were exposed to A) normal, B) drought and C) cold conditions. Fornormal conditions, plants were kept on a growth chamber set up to 22°C., 60% relative humidity and 14 h light/10 dark cycle for 15 days afterplanting. For drought, water was removed from bottom container indouble-decker Magenta box one week after planting and the sand was letto dry. Harvesting was done at 7 days after water was removed, whenwilting symptoms appeared. For cold, the air temperature was set to 5°C., one week after planting and maintained for 7 days. The roots of theinoculated plant are not only larger but also show a larger amount oflateral roots and root-hairs.

FIG. 7 is a graph depicting that seed-origin microbes show beneficialeffects across a wide range of administered doses. Sterilized wheatseeds were inoculated with 3.0×104, 3.0×105 and 3.0×106 CFU/seed ofendophytic microbes SYM00011, SYM00033 and SYM00057B. Shown are rootlengths of each treatment, represented as a percentage increase overmock-inoculated controls.

FIG. 8 contains three graphs depicting the field testing of seed-originmicrobes for benefits to cereal crop emergence. Top panel: Number ofwheat plants emerging in the middle 10′ section of the middle 2 rows ofeach test plot. Numbers reported are an average of counts of 6 replicateplots for each treatment. All SYM strains show improvement in emergenceover the untreated control. Middle panel: Improvement in the number ofcorn plants emerging in the dryland test plots over the untreatedcontrol. Emergence numbers were calculated as an average of counts of 6replicate plots for each treatment. All SYM strains show improvement inemergence over the untreated control, with SYM00260, SYM00290 andSYM00254 all showing improvements greater than 15%. Bottom panel:Improvement in the number of corn plants emerged in the irrigated testplots over the untreated control. Emergence numbers were calculated asan average of counts of 6 replicate plots for each treatment. All SYMstrains show improvement in emergence over the untreated control, withSYM00292 showing an improvement of 15%.

DEFINITIONS

An “endophyte” or “endophytic microbe” is an organism that lives withina plant or is otherwise associated therewith. Endophytes can occupy theintracellular or extracellular spaces of plant tissue, including theleaves, stems, flowers, fruits, seeds, or roots. An endophyte can beeither a bacterial or a fungal organism that can confer a beneficialproperty to a plant such as an increase in yield, biomass, resistance,or fitness in its host plant. As used herein, the term “microbe” issometimes used to describe an endophyte.

In some embodiments, a bacterial endophyte is a seed-origin bacterialendophyte. As used herein, a “seed-origin bacterial endophyte” refers toa population of bacteria associated with or derived from the seed of agrass plant. For example, a seed-origin bacterial endophyte can be foundin mature, dry, undamaged (e.g., no cracks, visible fungal infection, orprematurely germinated) seeds. The bacteria can be associated with orderived from the surface of the seed; alternatively, or in addition, itcan be associated with or derived from the interior seed compartment(e.g., of a surface-sterilized seed). In some cases, a seed-originbacterial endophyte is capable of replicating within the plant tissue,for example, the interior of the seed. Also, in some cases, theseed-origin bacterial endophyte is capable of surviving desiccation.

Seed-origin means that the bacterial entity is obtained directly orindirectly from the seed surface or seed interior compartment or isobtainable from a seed surface or seed interior compartment. Forexample, a seed-origin bacterial entity can be obtained directly orindirectly from a seed surface or seed interior compartment when it isisolated, or isolated and purified, from a seed preparation; in somecases, the seed-origin bacterial entity which has been isolated, orisolated and purified, may be cultured under appropriate conditions toproduce a purified bacterial population consisting essentially of aseed-origin bacterial endophyte. A seed-origin bacterial endophyte canbe considered to be obtainable from a seed surface or seed interiorcompartment if the bacteria can be detected on or in, or isolated from,a seed surface or seed interior compartment of a plant.

The compositions provided herein are preferably stable. The seed-originbacterial endophyte is optionally shelf stable, where at least 10% ofthe CFUs are viable after storage in desiccated form (i.e., moisturecontent of 30% or less) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greaterthan 10 weeks at 4° C. or at room temperature. Optionally, a shelfstable formulation is in a dry formulation, a powder formulation, or alyophilized formulation. In some embodiments, the formulation isformulated to provide stability for the population of bacterialendophytes. In one embodiment, the formulation is substantially stableat temperatures between about 0° C. and about 50° C. for at least about1, 2, 3, 4, 5, or 6 days, or 1, 2, 3 or 4 weeks, or 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11 or 12 months, or one or more years. In another embodiment,the formulation is substantially stable at temperatures between about 4°C. and about 37° C. for at least about 5, 10, 15, 20, 25, 30 or greaterthan 30 days.

An agricultural plant can be a monocotyledonous (i.e., an “agriculturalgrass plant”) or a dicotyledonous plant typically used in agriculture.An agricultural grass plant includes, but is not limited to, maize (Zeamays), common wheat (Triticum aestivum), spelt (Triticum spelta),einkorn wheat (Triticum monococcum), emmer wheat (Triticum dicoccum),durum wheat (Triticum durum), Asian rice (Oryza sativa), African rice(Oryza glabaerreima), wild rice (Zizania aquatica, Zizania latifolia,Zizania palustris, Zizania texana), barley (Hordeum vulgare), Sorghum(Sorghum bicolor), Finger millet (Eleusine coracana), Proso millet(Panicum miliaceum), Pearl millet (Pennisetum glaucum), Foxtail millet(Setaria italic), Oat (Avena sativa), Triticale (Triticosecale), rye(Secale cereal), Russian wild rye (Psathyrostachys juncea), bamboo(Bambuseae), or sugarcane (e.g., Saccharum arundinaceum, Saccharumbarberi, Saccharum bengalense, Saccharum edule, Saccharum munja,Saccharum officinarum, Saccharum procerum, Saccharum ravennae, Saccharumrobustum, Saccharum sinense, or Saccharum spontaneum).

A “host plant” includes any plant, particularly an agricultural plant,which an endophytic microbe such as a seed-origin bacterial endophytecan colonize. As used herein, a microbe is said to “colonize” a plant orseed when it can be stably detected within the plant or seed over aperiod time, such as one or more days, weeks, months or years; in otherwords, a colonizing microbe is not transiently associated with the plantor seed. A preferred host plant is a cereal plant.

As used herein, a “reference agricultural plant” is an agriculturalplant of the same species, strain, or cultivar to which a treatment,formulation, composition or endophyte preparation as described herein isnot administered/contacted. Exemplary reference agricultural plants arecereals. A reference agricultural plant, therefore, is identical to thetreated plant with the exception of the presence of the endophyte andcan serve as a control for detecting the effects of the endophyte thatis conferred to the plant.

“Biomass” means the total mass or weight (fresh or dry), at a giventime, of a plant tissue, plant tissues, an entire plant, or populationof plants. Biomass is usually given as weight per unit area. The termmay also refer to all the plants or species in the community (communitybiomass).

A “bacterial network” means a plurality of endophyte entities (e.g.,bacteria, fungi, or combinations thereof) co-localized in anenvironment, such as on or within a cereal agricultural plant.Preferably, a bacterial network includes two or more types of endophyteentities that synergistically interact, such synergistic endophyticpopulations capable of providing a benefit to the agricultural seed,seedling, or plant derived thereby.

An “increased yield” can refer to any increase in biomass or seed orfruit weight, seed size, seed number per plant, seed number per unitarea, bushels per acre, tons per acre, kilo per hectare, or carbohydrateyield. Typically, the particular characteristic is designated whenreferring to increased yield, e.g., increased grain yield or increasedseed size.

A “transgenic plant” includes a plant or progeny plant of any subsequentgeneration derived therefrom, wherein the DNA of the plant or progenythereof contains an exogenous DNA not naturally present in anon-transgenic plant of the same strain. The transgenic plant mayadditionally contain sequences that are native to the plant beingtransformed, but wherein the “exogenous” gene has been altered in orderto alter the level or pattern of expression of the gene, for example, byuse of one or more heterologous regulatory or other elements.

The terms “pathogen” and “pathogenic” in reference to a bacteriumincludes any such organism that is capable of causing or affecting adisease, disorder or condition of a host containing the organism.

A “spore” or a population of “spores” refers to bacteria that aregenerally viable, more resistant to environmental influences such asheat and bacteriocidal agents than vegetative forms of the samebacteria, and typically capable of germination and out-growth. Bacteriathat are “capable of forming spores” are those bacteria containing thegenes and other necessary abilities to produce spores under suitableenvironmental conditions.

As used herein, an “agricultural seed” is a seed used to grow a planttypically used in agriculture (an “agricultural plant”). The seed may beof a monocot or dicot plant, and may be planted for the production of anagricultural product, for example grain, food, fiber, etc. As usedherein, an agricultural seed is a seed that is prepared for planting,for example, in farms for growing.

In some cases, the present invention contemplates the use of microbesthat are “compatible” with agricultural chemicals, for example, afungicide, an anti-bacterial compound, or any other agent widely used inagricultural which has the effect of killing or otherwise interferingwith optimal growth of microbes. As used herein, a microbe is“compatible” with an agricultural chemical when the microbe is modified,such as by genetic modification, e.g., contains a transgene that confersresistance to an herbicide, or is adapted to grow in, or otherwisesurvive, the concentration of the agricultural chemical used inagriculture. For example, a microbe disposed on the surface of a seed iscompatible with the fungicide metalaxyl if it is able to survive theconcentrations that are applied on the seed surface.

In some embodiments, an agriculturally compatible carrier can be used toformulate an agricultural formulation or other composition that includesa purified bacterial preparation. As used herein an “agriculturallycompatible carrier” refers to any material, other than water, which canbe added to a seed or a seedling without causing or having an adverseeffect on the seed (e.g., reducing seed germination) or the plant thatgrows from the seed, or the like.

As used herein, a “portion” of a plant refers to any part of the plant,and can include distinct tissues and/or organs, and is usedinterchangeably with the term “tissue” throughout.

A “population” of plants, as used herein, can refer to a plurality ofplants that were subjected to the same inoculation methods describedherein, or a plurality of plants that are progeny of a plant or group ofplants that were subjected to the inoculation methods. In addition, apopulation of plants can be a group of plants that are grown from coatedseeds. The plants within a population will typically be of the samespecies, and will also typically share a common genetic derivation.

A “reference environment” refers to the environment, treatment orcondition of the plant in which a measurement is made. For example,production of a compound in a plant associated with a purified bacterialpopulation (e.g., a seed-origin bacterial endophyte) can be measured ina reference environment of drought stress, and compared with the levelsof the compound in a reference agricultural plant under the sameconditions of drought stress. Alternatively, the levels of a compound inplant associated with a purified bacterial population (e.g., aseed-origin bacterial endophyte) and reference agricultural plant can bemeasured under identical conditions of no stress.

As used herein, a nucleic acid has “homology” or is “homologous” to asecond nucleic acid if the nucleic acid sequence has a similar sequenceto the second nucleic acid sequence. The terms “identity,” “percentsequence identity” or “identical” in the context of nucleic acidsequences refer to the residues in the two sequences that are the samewhen aligned for maximum correspondence. There are a number of differentalgorithms known in the art that can be used to measure nucleotidesequence identity. For instance, polynucleotide sequences can becompared using FASTA, Gap or Bestfit, which are programs in WisconsinPackage Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTAprovides alignments and percent sequence identity of the regions of thebest overlap between the query and search sequences. Pearson, MethodsEnzymol. 183:63-98 (1990). The term “substantial homology” or“substantial similarity,” when referring to a nucleic acid or fragmentthereof, indicates that, when optimally aligned with appropriatenucleotide insertions or deletions with another nucleic acid (or itscomplementary strand), there is nucleotide sequence identity in at leastabout 76%, 80%, 85%, or at least about 90%, or at least about 95%, 96%,97%, 98% 99%, 99.5% or 100% of the nucleotide bases, as measured by anywell-known algorithm of sequence identity, such as FASTA, BLAST or Gap,as discussed above.

As used herein, the terms “operational taxon unit,” “OTU,” “taxon,”“hierarchical cluster,” and “cluster” are used interchangeably. Anoperational taxon unit (OTU) refers to a group of one or more organismsthat comprises a node in a clustering tree. The level of a cluster isdetermined by its hierarchical order. In one embodiment, an OTU is agroup tentatively assumed to be a valid taxon for purposes ofphylogenetic analysis. In another embodiment, an OTU is any of theextant taxonomic units under study. In yet another embodiment, an OTU isgiven a name and a rank. For example, an OTU can represent a domain, asub-domain, a kingdom, a sub-kingdom, a phylum, a sub-phylum, a class, asub-class, an order, a sub-order, a family, a subfamily, a genus, asubgenus, or a species. In some embodiments, OTUs can represent one ormore organisms from the kingdoms eubacteria, protista, or fungi at anylevel of a hierarchal order. In some embodiments, an OTU represents aprokaryotic or fungal order.

As used herein, a “colony-forming unit” (“CFU”) is used as a measure ofviable microorganisms in a sample. A CFU is an individual viable cellcapable of forming on a solid medium a visible colony whose individualcells are derived by cell division from one parental cell.

DETAILED DESCRIPTION

As demonstrated herein, agricultural plants, in particular cereals,appear to associate with symbiotic microorganisms termed endophytes,particularly bacteria and fungi, that may have been important duringevolution and may contribute to plant survival and performance. However,modern agricultural processes may have perturbed this relationship,resulting in increased crop losses, diminished stress resilience,biodiversity losses, and increasing dependence on external chemicals,fertilizers, and other unsustainable agricultural practices. There is aneed for novel methods for generating plants with novel microbiomeproperties that can sustainably increase yield, stress resilience, anddecrease fertilizer and chemical use.

Currently, the generally accepted view of plant endophytic communitiesfocuses on their homologous derivation, predominantly from the soilcommunities in which the plants are grown (Hallman, J., et al., (1997)Canadian Journal of Microbiology. 43(10): 895-914). Upon observingtaxonomic overlap between the endophytic and soil microbiota in A.thaliana, it was stated, “Our rigorous definition of an endophyticcompartment microbiome should facilitate controlled dissection ofplant-microbe interactions derived from complex soil communities”(Lundberg et al., (2012) Nature. 488, 86-90). There is strong support inthe art for soil representing the repository from which plant endophytesare derived. New Phytologist (2010) 185: 554-567. Notable plant-microbeinteractions such as mycorrhyzal fungi and bacterial rhizobia fit theparadigm of soil-based colonization of plant hosts and appear toprimarily establish themselves independently of seed. As a result offocusing attention on the derivation of endophytes from the soil inwhich the target agricultural plant is currently growing, there has beenan inability to achieve commercially significant improvements in plantyields and other plant characteristics such as increased root biomass,increased root length, increased height, increased shoot length,increased leaf number, increased water use efficiency, increased overallbiomass, increase grain yield, increased photosynthesis rate, increasedtolerance to drought, increased heat tolerance, increased salttolerance, increased resistance to nematode stress, increased resistanceto a fungal pathogen, increased resistance to a bacterial pathogen,increased resistance to a viral pathogen, a detectable modulation in thelevel of a metabolite, and a detectable modulation in the proteomerelative to a reference plant.

In part, the present invention describes preparations of novelseed-derived endophytes, and the creation of synthetic combinations ofcereal seeds and/or seedlings with heterologous seed-derived endophytesand formulations containing the synthetic combinations, as well as therecognition that such synthetic combinations display a diversity ofbeneficial properties present in the agricultural plants and theassociated endophyte populations newly created by the present inventors.Such beneficial properties include metabolism, transcription, proteomealterations, morphology, and the resilience to a variety ofenvironmental stresses, and the combination of a plurality of suchproperties.

Little attention has been provided in the art to understand the role ofseeds as reservoirs for microbes that can efficiently populate theendosphere of cereal plants. While the concept that seeds may harborplant pathogens was promoted by Baker and Smith (Annu Rev Phytopathol14: 311-334(1966)), and the understanding that bacterial and fungalpathogens are known to be able to infect seed, the ability to harnessendophytes derived from a broad spectrum of seeds to heterologouslyconfer single or multiple advantages to cereal crops was previouslyunrecognized. As the presence of detectable pathogens in a seed lot cannecessitate destruction of vast numbers of agricultural germplasm(Gitaitis, R. and Walcott, R. (2007) Annu. Rev. Phytopathol. 45:371-97),safety concerns have surrounded the consideration of seed-associatedmicrobes or non-soil endophytes. Moreover, when seed pathogens aredetected, their transfer to the growing plant can be highly inefficient.For example, a study of seed-based transmission of the seed pathogen,Pantoea stewartii, found that seed produced from a population ofpathogen-infected plants gave rise to infected seedlings in only 0.0029%of cases (1 of 34,924 plants) and artificially infected kernels onlygave rise to infected seedlings in 0.022% of cases (Block, C. C., etal., (1998). Plant disease. 82(7). 775-780). Thus, the efficiency withwhich plants introduce microbes into their seeds, and with whichmicrobes within seeds propagate within the resulting plant tissues, hasbeen previously thought to be low and often substantially variable.Thus, the potential for microbial content within cereal seeds topopulate the resulting plant has been unclear.

The potential for agricultural cereal seeds to serve as reservoirs fornon-pathogenic microbes also remains controversial (Hallman, J., et al.,(1997) Canadian Journal of Microbiology. 43(10): 895-914). Sato, et al.,did not detect any bacteria inside rice seeds ((2003) In. Morishima, H.(ed.) The Natural History of Wild Rice—Evolution Ecology of Crop. p.91-106) and Mundt and Hinkle only obtained endophytes from seed sampleswhere seed coats had been broken or fractured in 29 kinds of plant seed(Appl Environ Microbiol. (1976) 32(5):694-8). Another group detectedsimply bacterial populations inside rice seeds ranging in populationsize from 10̂2 to 10̂6 CFU/g fresh weight (Okunishi, S., et al., (2005)Microbes and Environment. 20:168-177). Rosenblueth et al described seedsto harbor very simple microbial communities with significant variabilityof the microbial communities between individual maize seeds, includingsubstantial variability between seeds taken from the same cobb(Rosenblueth, M. et al, Seed Bacterial Endophytes: Common Genera,Seed-to-Seed Variability and Their Possible Role in Plants; Proc.XXVIIIth IHC—IS on Envtl., Edaphic & Gen. Factors; Affecting Plants,Seeds and Turfgrass; Eds.: G. E. Welbaum et al. Acta Hort. 938, ISHS2012).

These findings demonstrate limitations recognized in the art regardingthe attempted use of endophytes derived from seeds; i.e., maize seedsappear to contain limited taxonomic diversity, and that the microbiotaof individual seeds produced by plants is often distinct, indicatingthat there may not be single seed-derived symbionts capable of providingbenefits across a large population of agricultural plants and inspecific, the utilization of endophytes on seed. For example,characterization of ˜15 pooled seeds from within various cultivars fromthe genus Zea showed that populations of maize seeds tend to harbor avery limited number of taxa that appear to be conserved across modernand ancestral variants, and that the maize seed content of such taxa islow and substantially variable. It is unclear whether the presence ofsuch limited taxa resulted from common storage conditions, environmentalcontamination, or a potential vertical transmission of microbes viaseeds, and also uncertain was the applicability of such limited taxa inincreasing agricultural yield. Notably, 99% of these strains were shownto provide detrimental or to lack beneficial effects on agriculturalplants, e.g., when tested in a potato growth assay (i.e., a non-cerealcrop) (Johnston-Monje D, Raizada M N (2011) Conservation and Diversityof Seed Associated Endophytes in Zea across Boundaries of Evolution,Ethnography and Ecology. PLoS ONE 6(6): e20396.doi:10.1371/journal.pone.0020396). Further, some of the microbesisolated bear close evolutionary relation to plant pathogens, making itpossible that such microbes represent a latent reservoir of pathogens,rather than potentially beneficial constituents.

Surprisingly, it was discovered here that seed-derived endophytes canconfer significant advantages to cereal crops, spanning growth undernormal and stressed conditions, altered expression of key planthormones, altered expression of key transcripts in the plant, and otherdesirable features. Provided are novel compositions, methods, andproducts related our invention's ability to overcome the limitations ofthe prior art in order to provide reliable increases in cereal yield,biomass, germination, vigor, stress resilience, and other properties toagricultural crops.

The invention described herein is surprising for multiple reasons basedon the previous demonstrations in the art. Notably, there is a lack ofclarity related to whether endophytes are associated with healthy cerealseeds, whether microbes isolated from cereal seeds could efficientlycolonize the cereal host if disposed on the exterior of a seed orseedling, and whether such microbes would confer a beneficial ordetrimental effects on cereal hosts. It is further unclear whether theheterologous application of such microbes to distinct cereal seeds fromwhich they were derived could provide beneficial effects.

As described herein, beneficial microbes can be robustly derived fromagricultural seeds, optionally cultured, administered heterologously toagricultural cereal seeds or seedlings, and colonize the resulting planttissues with high efficiency to confer multiple beneficial properties.This is surprising given the variability observed in the art in microbeisolation from healthy seeds and the previous observations ofinefficient seed pathogen colonization of plant host's tissues. Further,the ability of heterologously disposed seed-derived endophytes tocolonize seeds and seedlings from the exterior of seeds is surprising,given that such endophytes can be isolated from within internal seedtissues and therefore do not natively need the capacity to externallypenetrate and invade into host tissues.

Prior characterization of microbial content of seeds has indicated thatmicrobial concentrations in seeds can be variable and are generally verylow (i.e., less than 10, 100, 10³, 10⁴, 10⁵ CFUs/seed). As such, it wasunclear whether altered or increased concentrations of microbesassociated with seeds could be beneficial. We find that microbes canconfer beneficial properties across a range of concentrations.

We find that seed-derived endophytes can be heterologously disposed ontoseedlings of a distinct cultivar, species, or cereal crop type andconfer benefits to those new recipients. For example, seed-derivedendophytes from corn cultivars are heterologously provided to wheatcultivars to confer a benefit. This is surprising given the observationsof distinct microbiome preferences in distinct plant and mammalian hostsand, in particular, the likelihood that microbes derived from seeds havebeen co-evolved to be specialized to a particular host.

We further find that combinations of heterologously disposedseed-derived endophytes confer additive advantages to plants, includingmultiple functional properties and resulting in seed, seedling, andplant hosts that display single or multiple improved agronomicproperties.

In general, this application provides methods and materials forproviding a benefit to a seed or seedling of an agricultural grass plantusing purified bacterial populations that include novel seed-originendophytes that are unique in that they have been isolated from seeds ofgrass plants. Such seed-origin bacterial endophytes can providebeneficial properties to the seed, seedling, or the agricultural grassplant derived from the seed or seedling, including benefits tometabolism, transcription, proteome alterations, morphology, and theresilience to a variety of environmental stresses, and combinations ofsuch properties.

As described herein, synthetic combinations that include a host plantsuch as an agricultural grass plant associated with a purified bacterialpopulation that contains an endophyte, e.g., a seed-origin bacterialendophyte can be used to provide the benefit to a seed, seedling, oragricultural plant derived from the seed or seedling. The syntheticcombination may be produced, for example, by inoculation, application tofoliage (e.g., by spraying) or to seeds (e.g., coating of seeds),grafting, root dips, soil drenches, or infection of a host plant, hostplant tissues, or a seed, or combinations thereof, as described herein.In any of the methods, any of such techniques can be used to makesynthetic combinations. Inoculation, application to foliage or seeds, orinfection can be particularly useful.

In some embodiments, the invention uses microbes that are heterologousto a seed or plant in making synthetic combinations or agriculturalformulations. A microbe is considered heterologous to the seed or plantif the seed or seedling that is unmodified (e.g., a seed or seedlingthat is not treated with a bacterial endophyte population describedherein) does not contain detectable levels of the microbe. For example,the invention contemplates the synthetic combinations of seeds orseedlings of agricultural plants (e.g., agricultural grass plants) andan endophytic microbe population (e.g., a seed-origin bacterialendophyte), in which the microbe population is “heterologously disposed”on the exterior surface of or within a tissue of the agricultural seedor seedling in an amount effective to colonize the plant. A microbe isconsidered “heterologously disposed” on the surface or within a plant(or tissue) when the microbe is applied or disposed on the plant in anumber that is not found on that plant before application of themicrobe. For example, a bacterial endophytic population that is disposedon an exterior surface or within the seed can be an endophytic bacteriumthat may be associated with the mature plant, but is not found on thesurface of or within the seed. As such, a microbe is deemedheterologously disposed when applied on the plant that either does notnaturally have the microbe on its surface or within the particulartissue to which the microbe is disposed, or does not naturally have themicrobe on its surface or within the particular tissue in the numberthat is being applied. Indeed, several of the endophytic microbesdescribed herein have not been detected, for example, in any of the cornseeds sampled, as determined by highly sensitive methods.

In some embodiments, a microbe can be “endogenous” to a seed or plant.As used herein, a microbe is considered “endogenous” to a plant or seed,if the microbe is derived from, or is otherwise found in, the seed orthe plant, or any plant or seed of the same species. In embodiments inwhich an endogenous microbe is applied, the endogenous microbe isapplied in an amount that differs from the levels typically found in theplant.

Seed-Origin Bacterial Endophytes

In some embodiments, this application relates to purified bacterialpopulations that contain seed-origin bacterial endophytes from, forexample, maize, wheat, rice, or barley, compositions such asagricultural formulations or articles of manufacture that include suchpurified bacterial populations, as well as methods of using suchbacterial populations to make synthetic combinations or agriculturalproducts. A seed-origin bacterial endophyte used in a composition orused to make a synthetic composition can be obtained from the samecultivar or species of agricultural plant to which the composition isbeing applied or can be obtained from a different cultivar or species ofagricultural plant.

Many bacterial species are sensitive to conditions of drying anddesiccation. Surprisingly, the bacterial endophytes described hereinhave been isolated from mature, dry seeds of grass plants, includingmaize, rice, and wheat seeds. The recovery of viable bacterialendophytes from these mature dry seeds demonstrates that, unlike mostother bacteria, these seed-origin bacterial endophytes are capable ofsurviving conditions of desiccation. Therefore, in one embodiment, thepurified bacterial population containing seed-origin bacterialendophytes is desiccation tolerant. For example, a substantial portionof the population (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or greater than 99%)of the seed-origin bacterial endophytes can survive in moisture contentlevels of 30% or less, for example, 25% or less, 20% or less, 15% orless, 12% or less, 10% or less, or 8% or less, for a period of at least1 day, for example, at least 3 days, at least 5 days, at least 7 days,at least 10 days, at least 14 days, at least 21 days, at least 30 days,at least 45 days, at least 60 days, or more, within the seeds of a grassplant that are stored at between 1° C. and 35° C.

In another embodiment, the seed-origin bacterial endophyte is capable offorming spores. In still another embodiment, at least 1% of thepopulation of the seed-origin bacterial endophyte, for example, at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 75%, at least 80%, at least 90%, or at least 95% or more,is used in spore form.

In some embodiments, the seed-origin bacterial endophyte can be culturedon a culture medium or can be adapted to culture on a culture medium.

In some embodiments, a purified bacterial population is used thatincludes two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 orgreater than 25) different seed-origin bacterial endophytes, e.g.,obtained from different families or different genera of bacteria, orfrom the same genera but different species of bacteria. The differentseed-origin bacterial endophytes can be obtained from the same cultivarof agricultural plant (e.g., the same maize, wheat, rice, or barleyplant), different cultivars of the same agricultural plant (e.g., two ormore cultivars of maize, two or more cultivars of wheat, two or morecultivars of rice, or two or more cultivars of barley), or differentspecies of the same type of agricultural plant (e.g., two or moredifferent species of maize, two or more different species of wheat, twoor more different species of rice, or two or more different species ofbarley). In embodiments in which two or more seed-origin bacterialendophytes are used, each of the seed-origin bacterial endophytes canhave different properties or activities, e.g., produce differentmetabolites, produce different enzymes such as different hydrolyticenzymes, confer different beneficial traits, or colonize different partsof a plant (e.g., leaves, stems, flowers, fruits, seeds, or roots). Forexample, one seed-origin bacterial endophyte can colonize a first and asecond seed-origin bacterial endophyte can colonize a tissue thatdiffers from the first tissue. Combinations of bacterial endophytes arediscussed in detail below.

In one embodiment, the endophyte is an endophytic microbe isolated froma different plant than the inoculated plant. For example, in oneembodiment, the endophyte is an endophyte isolated from a differentplant of the same species as the inoculated plant. In some cases, theendophyte is isolated from a species related to the inoculated plant.

The breeding of plants for agriculture, as well as cultural practicesused to combat microbial pathogens, may have resulted in the loss inmodern cultivars of the endophytes present in their wild ancestors, orsuch practices may have inadvertently promoted other novel or rareplant-endophyte interactions, or otherwise altered the microbialpopulation. We hypothesized that an altered diversity and titer ofendophytes in the ancestor could correlate with an altered range ofphysiological responses derived from the symbiosis that allow the plantto better adapt to the environment and tolerate stress. In order tosurvey plant groups for potentially useful endophytes, seeds of theirwild ancestors, wild relatives, primitive landraces, modern landraces,modern breeding lines, and elite modern agronomic varieties are screenedfor microbial endophytes by culture and culture independent methods asdescribed herein.

In some cases, plants are inoculated with endophytes that areheterologous to the seed of the inoculated plant. In one embodiment, theendophyte is derived from a plant of another species. For example, anendophyte that is normally found in dicots is applied to a monocot plant(e.g., inoculating corn with a soy bean-derived endophyte), or viceversa. In other cases, the endophyte to be inoculated onto a plant isderived from a related species of the plant that is being inoculated. Inone embodiment, the endophyte is derived from a related taxon, forexample, from a related species. The plant of another species can be anagricultural plant. For example, an endophyte derived from Hordeumirregulare can be used to inoculate a Hordeum vulgare L., plant.Alternatively, it is derived from a ‘wild’ plant (i.e., anon-agricultural plant). For example, endophytes normally associatedwith the wild cotton Gossypium klotzschianum are useful to inoculatecommercial varieties of Gossypium hirsutum plants. As an alternativeexample of deriving an endophyte from a ‘wild’ plant, endophyticbacteria isolated from the South East Asian jungle orchid, Cymbidiumeburneum, can be isolated and testing for their capacity to benefitseedling development and survival of agricultural crops such as wheat,maize, soy and others (Faria, D. C., et al., (2013) World Journal ofMicrobiology and Biotechnology. 29(2). pp. 217-221). In other cases, theendophyte can be isolated from an ancestral species of the inoculatedplant. For example, an endophyte derived from Zea diploperennis can beused to inoculate a commercial variety of modern corn, or Zea mays.

In some embodiments, a purified bacterial populations containsseed-origin bacterial endophytes from one or more (e.g., two, three,four, five, six, seven, eight, nine, 10, or more families selected fromthe group consisting of Acidithiobacillaceae, Actinosynnemataceae,Aerococcaceae, Aeromonadaceae, Alcaligenaceae, Alteromonadaceae,Bacillaceae, Bdellovibrionaceae, Bradyrhizobiaceae, Brucellaceae,Burkholderiaceae, Carnobacteriaceae, Caulobacteraceae,Cellulomonadaceae, Chitinophagaceae, Chromatiaceae, Clostridiaceae,Comamonadaceae, Coriobacteriaceae, Corynebacteriaceae, Deinococcaceae,Ectothiorhodospiraceae, Enterobacteriaceae, Flavobacteriaceae,Halomonadaceae, Hyphomicrobiaceae, Lachnospiraceae, Lactobacillaceae,Methylobacteriaceae, Microbacteriaceae, Micrococcaceae, Moraxellaceae,Mycobacteriaceae, Neisseriaceae, Nocardiaceae, Oxalobacteraceae,Paenibacillaceae, Planococcaceae, Propionibacteriaceae,Pseudonocardiaceae, Rhizobiaceae, Rhodospirillaceae,Sphingobacteriaceae, Sphingomonadaceae, Streptomycetaceae,Tissierellaceae, Weeksellaceae, Xanthobacteraceae, and Xanthomonadaceae.

In one embodiment, the purified bacterial population includesseed-origin bacterial endophytes is from one or more families selectedfrom the group consisting of Xanthomonadaceae, Sphingomonadaceae,Weeksellaceae, Microbacteriaceae, Micrococcaceae, Methylobacteriaceae,Xanthomonadaceae, Rhizobiaceae, Paenibacillaceae, Staphylococcaceae,Enterobacteriaceae, Pseudomonadaceae, and Bacillaceae.

In some embodiments, the purified bacterial population includesseed-origin bacterial endophytes from one or more (e.g., two, three,four, five, six, seven, eight, nine, 10, or more) of the generasselected from the group consisting of Achromobacter, Acidithiobacillus,Acidovorax, Acidovoraz, Acinetobacter, Aerococcus, Aeromonas, Agromyces,Ancylobacter, Arthrobacter, Azospirillum, Bacillus, Bdellovibrio, Bosea,Bradyrhizobium, Brevibacillus, Brevundimonas, Burkholderia,Cellulomonas, Cellvibrio, Chryseobacterium, Citrobacter, Clostridium,Corynebacterium, Cupriavidus, Curtobacterium, Curvibacter, Deinococcus,Desemzia, Devosia, Dokdonella, Dyella, Enhydrobacter, Enterobacter,Enterococcus, Erwinia, Escherichia, Finegoldia, Flavisolibacter,Flavobacterium, Frigoribacterium, Hafnia, Halomonas, Herbaspirillum,Klebsiella, Kocuria, Lactobacillus, Leclercia, Lentzea, Luteibacter,Luteimonas, Massilia, Methylobacterium, Microbacterium, Micrococcus,Microvirga, Mycobacterium, Neisseria, Nocardia, Oceanibaculum,Ochrobactrum, Oxalophagus, Paenibacillus, Panteoa, Pantoea,Plantibacter, Propionibacterium, Propioniciclava, Pseudomonas,Pseudonocardia, Pseudoxanthomonas, Psychrobacter, Rheinheimera,Rhizobium, Rhodococcus, Roseateles, Ruminococcus, Sediminibacillus,Sediminibacterium, Serratia, Shigella, Shinella, Sphingobacterium,Sphingomonas, Sphingopyxis, Sphingosinicella, Staphylococcus,Stenotrophomonas, Strenotrophomonas, Streptomyces, Tatumella,Tepidimonas, Thermomonas, Thiobacillus, Uncultured bacterium,Variovorax, and Xanthomonas.

In some embodiments, the purified bacterial population does not includeat least one of Acetobacter sp., Acidovorax facilis, Azospirillumbrasilense, Azospirillum hpoferum, Azospirillum sp., Azotobacter sp.,Azotobacter vinelandii, Bacillus amyloliquefaciens FZB42, Bacillusamyloliquefaciens strain D747, Bacillus amyloliquefaciens TJ1000,Bacillus amyloliquefaciens TM45, Bacillus chitinosporus, Bacillusfirmus, Bacillus firmus NCIM 2637, Bacillus firmus 1-1582, Bacilluslaterosporus, Bacillus licheniformis, Bacillus licheniformus, Bacillusmarinus, Bacillus megaterium, Bacillus megaterium var. phosphaticum,Bacillus megatherium, Bacillus oleronius, Bacillus pumilus, Bacilluspumilus QST 2808, Bacillus sp., Bacillus subtilis, Bacillus subtilisFZB24, Bacillus subtilis MBI 600, Bacillus subtilis BSF4, Bacillussubtilis MBI600, Bacillus subtilis QST 713, Bacillus thuringensis varKurstaki (NCIM 2514), Bacillus thuringiensis aizawai, Bacillusthuringiensis kurstaki, Bacillus thuringiensis kurstaki strain EG7841,Bacillus thuringiensis kurstaki strain SA-11, Bacillus thuringiensissubsp. kurstaki ABTS-351, Bacillus thuringiensis SV kurstaki EG 2348,Bacillus thuringiensis var Israelensis, Bacillus thuringiensis, Kurstakivariety, serotype 3A 3B, Bacillus thuringiensis, subsp. aizawai, StrainABTS-1857, Bacillus thuringiensis, subsp. israelensis, strain AM 65-52,Chromobacterium subtsugae strain PRAA4-1, Delftia acidovorans, Frateuriaaurantia, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillusfermentum, Lactobacillus helveticus, Lactobacillus plantarum,Lactococcus lactus, Methylobacterium mesophilicum, Methylobacteriumorganophilum, Methylobacterium extorquens, Paenibacillus polymyxa,Pasteuria spp., Pseudomonas spp., Pseudomonas fluorescens, Rhizobiumsp., Rhodococcus rhodochrous, Rhodopseudomonas palustris, Streptomyceslydicus WYEC 108, Streptomyces ray, or Thiobacillus thiooxidans.

In some embodiments, the purified fungal population does not include atleast one of Acremonium butyri, Ampelomyces quisqualis, Ampelomycesquisqualis (DSM 2222), Ampelomyces quisqualis M-10, Arthrobotrysoligospora, Aspergillus oryzae, Beauvaria bassiana strain ATCC 74040,Beauveria bassiana, Beauveria bassiana (NCIM 1216 ATCC 26851), Beauveriabassiana strain GHA, Beauveria bassiana strain GHA 1991, Candida utilis,Chaetomium cupreum (CABI 353812), Chaetomium globosum, Clonostachysrosea 88-710, Fusarium oxysporum IF23, Fusarium proliferatum (NCIM1101), Gliocladium, Gliocladium catenulatum strain J1446, Gliocladiumvirens GL-21, Glomus fasciculatum, Glomus intraradices, Hirsutellarhossiliensis, Isaria fumosorosea Apopka Strain 97, Metarhiziumanisopliae, Metarhizium anisopliae (NCIM 1311), Metschnikowiafructicola, Myrothecium verrucaria, Neotyphodium lolii AR1, Neotyphodiumlolii AR37, Neotyphodium lolii AR6, Neotyphodium lolii NEA2,Neotyphodium uncinatum, Paecilomyces fumorosoroseus strain FE 9901,Paecilomyces fumosoroseus, Paecilomyces lilacinus, Paecilomyceslilacinus (IIHR PL-2), Penicillium bilaii, Saccharomyces cerevisiae,Sclerotinia minor, Trichoderma asperellum TV1, Trichoderma asperellumstrain ICC 012, Trichoderma gamsii strain ICC 080, Trichodermaharzianum, Trichoderma harzianum (IIHR-Th-2), Trichoderma harzianumRifai strain T22, Trichoderma koningii, Trichoderma lignorum,Trichoderma polysporum, Trichoderma sp., Trichoderma virens G1-3,Trichoderma viride, Trichoderma viride (TNAU), Verticillium lecanii, orVerticillium lecanii (NCIM 1312).

In some embodiments, the purified bacterial population includesseed-origin bacterial endophytes from a non-Bacillus and/or anon-Pseudomonas genera and/or a non-Rhizobium genera, e.g., from one ormore of Achromobacter, Acidithiobacillus, Acidovorax, Acidovoraz,Acinetobacter, Aerococcus, Aeromonas, Agromyces, Ancylobacter,Arthrobacter, Azospirillum, Bdellovibrio, Bosea, Bradyrhizobium,Brevibacillus, Brevundimonas, Burkholderia, Cellulomonas, Cellvibrio,Chryseobacterium, Citrobacter, Clostridium, Corynebacterium,Cupriavidus, Curtobacterium, Curvibacter, Deinococcus, Desemzia,Devosia, Dokdonella, Dyella, Enhydrobacter, Enterobacter, Enterococcus,Erwinia, Escherichia, Finegoldia, Flavisolibacter, Flavobacterium,Frigoribacterium, Hafnia, Halomonas, Herbaspirillum, Klebsiella,Kocuria, Lactobacillus, Leclercia, Lentzea, Luteibacter, Luteimonas,Massilia, Methylobacterium, Microbacterium, Micrococcus, Microvirga,Mycobacterium, Neisseria, Nocardia, Oceanibaculum, Ochrobactrum,Oxalophagus, Paenibacillus, Panteoa, Pantoea, Plantibacter,Propionibacterium, Propioniciclava, Pseudonocardia, Pseudoxanthomonas,Psychrobacter, Rheinheimera, Rhodococcus, Roseateles, Ruminococcus,Sediminibacillus, Sediminibacterium, Serratia, Shigella, Shinella,Sphingobacterium, Sphingomonas, Sphingopyxis, Sphingosinicella,Staphylococcus, Stenotrophomonas, Strenotrophomonas, Streptomyces,Tatumella, Tepidimonas, Thermomonas, Thiobacillus, Uncultured bacterium,Variovorax, or Xanthomonas.

In some embodiments, the purified bacterial population includesseed-origin bacterial endophytes from a genera selected from the groupconsisting of Luteibacter, Sphingobium, Chryseobacterium,Curtobacterium, Micrococcus, Sphingomonas, Microbacterium,Methylobacterium, Stenotrophomonas, Xanthomonas, Agrobacterium,Paenibacillus, Staphylococcus, Enterobacter, Pantoea, Pseudomonas, andBacillus. In some embodiments, the purified bacterial populationsincludes seed-origin bacterial endophytes from a non-Bacillus, and/or anon-Pseudomonas genera and/or a non-Rhizobium genera, e.g., from one ormore of Luteibacter, Sphingobium, Chryseobacterium, Curtobacterium,Micrococcus, Sphingomonas, Microbacterium, Methylobacterium,Stenotrophomonas, Xanthomonas, Agrobacterium, Paenibacillus,Staphylococcus, Enterobacter, or Pantoea.

In some embodiments, the seed-origin bacterial endophyte includes a 16Snucleic acid sequence that is at least 97% identical to at least one ofthe nucleic acid sequences referenced in Table 1 or Table 2 (SEQ ID NOs:1-1448, e.g., SEQ ID NOs: 521-1448). For example, the seed-originbacterial endophyte can include a 16S nucleic acid sequence that is atleast 98% identical, at least 99% identical, or at least 99.5% identicalto a 16S nucleic acid sequence referenced in Table 1 or Table 2 (SEQ IDNOs: 1-1448, e.g., SEQ ID NOs: 521-1448). In some embodiments, theseed-origin bacterial endophyte comprises a 16S nucleic acid sequencethat is 100% identical to a 16S nucleic acid sequence referenced inTable 1 or Table 2 (SEQ ID NOs: 1-1448, e.g., SEQ ID NOs: 521-1448). Inembodiments in which two or more seed-origin bacterial endophytes areused, the 16S nucleic acid sequence of each seed-origin bacterialendophyte can have more than 97% sequence identity to each other or canhave less than 97% sequence identity to each other. In addition, inembodiments in which two or more seed-origin bacterial endophytes areused, the 16S nucleic acid sequence of one seed-origin bacterialendophyte can have more than 97% sequence identity to one of thenucleotide sequences set forth in SEQ ID NOs: 1-1448, and oneseed-origin bacterial endophyte can have less than 97% sequence identityto one of the 16S nucleotide sequences set forth in SEQ ID NOs:1-1448.

In some embodiments, the seed-origin bacterial endophyte includes a 16Snucleic acid sequence that has less than 97% sequence identity to atleast one of the nucleic acid sequences referenced in Table 1 or Table 2(SEQ ID NOs: 1-1448).

TABLE 1 Representative endophytes from grass seeds, including their 16SrRNA sequences, assignment within OTU numbers, Genus, species, straininformation, as well as GenBank Accession numbers. SEQ ID NO. OTU #Genus Species Strain Accession No. 1 37 Burkholderia fungorum JF753273 237 Burkholderia fungorum JF753274 3 27 Burkholderia gladioli JF753275 421 Citrobacter freundii JF753276 5 21 Citrobacter freundii JF753277 6 61Clostridium acetobutylicum JF753278 7 61 Clostridium beijerinckiiJF753279 8 0 Enterobacter absuriae JF753280 9 0 Enterobacter absuriaeJF753281 10 0 Enterobacter aerogenes JF753282 11 0 Enterobacteraerogenes JF753283 12 18 Enterobacter asburiae JF753284 13 156Enterobacter asburiae JF753285 14 0 Enterobacter asburiae JF753286 15 0Enterobacter asburiae JF753287 16 0 Enterobacter asburiae JF753288 17 0Enterobacter asburiae JF753289 18 0 Enterobacter asburiae J2S4 JF75329019 18 Enterobacter asburiae MY2 JF753291 20 18 Enterobacter asburiae MY2JF753292 21 21 Enterobacter asburiae J2S4 JF753293 22 18 Enterobactercloacae JF753294 23 18 Enterobacter cloacae JF753295 24 177 Enterobacterludwigii AR1.22 JF753296 25 56 Enterobacter sp. Nj-68 JF753297 26 18Escherichia coli JF753298 27 18 Escherichia coli JF753299 28 18Escherichia coli JF753300 29 18 Escherichia coli NBRI1707 JF753301 30 18Escherichia coli NBRI1707 JF753302 31 18 Escherichia coli NBRI1707JF753303 32 18 Klebsiella pneumoniae 342 JF753304 33 82 Luteibacter sp.JF753305 34 81 Methylobacterium sp. JF753306 35 31 Paenibacilluscaespitis JF753307 36 49 Paenibacillus ruminocola G22 JF753308 37 18Panteoa agglomerans JF753309 38 109 Pantoea agglomerans JF753310 39 146Pantoea agglomerans JF753311 40 109 Pantoea agglomerans 1.2244 JF75331241 84 Pantoea agglomerans 1.2244 JF753313 42 0 Pantoea agglomerans1.2244 JF753314 43 0 Pantoea agglomerans 1.2244 JF753315 44 84 Pantoeaagglomerans 1.2244 JF753316 45 7 Pantoea agglomerans BJCP2 JF753317 4658 Pantoea agglomerans BJCP2 JF753318 47 146 Pantoea agglomerans KJPB2JF753319 48 164 Pantoea agglomerans KJPB2 JF753320 49 0 Pantoeaagglomerans Sc-1 JF753321 50 0 Pantoea agglomerans Sc-1 JF753322 51 0Pantoea agglomerans Sc-1 JF753323 52 0 Pantoea agglomerans Sc-1 JF75332453 0 Pantoea agglomerans Sc-1 JF753325 54 0 Pantoea agglomerans Sc-1JF753326 55 0 Pantoea agglomerans Sc-1 JF753327 56 0 Pantoea agglomeransSc-1 JF753328 57 0 Pantoea agglomerans Sc-1 JF753329 58 0 Pantoeaagglomerans Sc-1 JF753330 59 0 Pantoea agglomerans Sc-1 JF753331 60 0Pantoea agglomerans Sc-1 JF753332 61 0 Pantoea agglomerans Sc-1 JF75333362 164 Pantoea agglomerans TX4CB_114 JF753334 63 0 Pantoea agglomeransTX4CB_114 JF753335 64 164 Pantoea agglomerans 1.2244 JF753336 65 84Pantoea agglomerans 1.2244 JF753337 66 84 Pantoea agglomerans 1.2244JF753338 67 84 Pantoea agglomerans 1.2244 JF753339 68 84 Pantoeaagglomerans 1.2244 JF753340 69 0 Pantoea agglomerans 48b/90 JF753341 70127 Pantoea agglomerans 48b/90 JF753342 71 0 Pantoea agglomerans 48b/90JF753343 72 7 Pantoea agglomerans BJCP2 JF753344 73 7 Pantoeaagglomerans BJCP2 JF753345 74 0 Pantoea agglomerans AN3 JF753346 75 84Pantoea agglomerans KJPB2 JF753347 76 164 Pantoea agglomerans KJPB2JF753348 77 84 Pantoea agglomerans KJPB2 JF753349 78 164 Pantoeaagglomerans KJPB2 JF753350 79 0 Pantoea agglomerans KJPB2 JF753351 80 0Pantoea agglomerans new*47con JF753352 81 0 Pantoea agglomerans Sc-1JF753353 82 0 Pantoea agglomerans Sc-1 JF753354 83 0 Pantoea agglomeransSc-1 JF753355 84 0 Pantoea agglomerans Sc-1 JF753356 85 0 Pantoeaagglomerans Sc-1 JF753357 86 173 Pantoea agglomerans Sc-1 JF753358 87199 Pantoea ananatis LMG 20103 JF753359 88 0 Pantoea ananatis LMG 20103JF753360 89 0 Pantoea ananatis LMG 20103 JF753361 90 0 Pantoea ananatisLMG 20103 JF753362 91 0 Pantoea ananatis LMG 20103 JF753363 92 0 Pantoeaananatis LMG 20103 JF753364 93 0 Pantoea ananatis LMG 20103 JF753365 940 Pantoea ananatis LMG 20103 JF753366 95 0 Pantoea ananatis LMG 20106JF753367 96 158 Pantoea ananatis SK-1 JF753368 97 0 Pantoea ananatisSK-1 JF753369 98 0 Pantoea sp. GJT-8 JF753370 99 0 Pantoea sp. GJT-8JF753371 100 0 Pantoea sp. GJT-8 JF753372 101 0 Pantoea sp. GJT-8JF753373 102 0 Pantoea sp. GJT-8 JF753374 103 0 Pantoea sp. GJT-8JF753375 104 0 Pantoea sp. GJT-8 JF753376 105 0 Pantoea sp. GJT-8JF753377 106 0 Pantoea sp. GJT-8 JF753378 107 0 Pantoea sp. GJT-8JF753379 108 1 Pseudomonas fluorescens JF753380 109 2 Pseudomonasoleovarans JF753381 110 2 Pseudomonas oryzihabitans JF753382 111 10Strenotrophomonas maltophilia JF753383 112 105 Strenotrophomonasmaltophilia JF753384 113 40 Strenotrophomonas maltophilia JF753385 11410 Strenotrophomonas maltophilia JF753386 115 10 Strenotrophomonasmaltophilia JF753387 116 185 Strenotrophomonas maltophilia JF753388 11710 Strenotrophomonas maltophilia JF753389 118 10 Strenotrophomonasmaltophilia JF753390 119 10 Strenotrophomonas maltophilia JF753391 12010 Strenotrophomonas maltophilia JF753392 121 10 Strenotrophomonasmaltophilia JF753393 122 10 Strenotrophomonas maltophilia JF753394 12310 Strenotrophomonas maltophilia JF753395 124 153 Strenotrophomonasmaltophilia JF753396 125 10 Strenotrophomonas maltophilia JF753397 12610 Strenotrophomonas maltophilia JF753398 127 86 Uncultured bacteriumUncultured bacterium SP6-0 JF753399 128 188 Uncultured bacteriumUncultured bacterium X-50 JF753400 129 84 Pantoea agglomerans 1.2244JF753401 130 179 Rhodococcus fascians JF753402 131 2 Pseudomonasoryzihabitans JF753403 132 84 Pantoea agglomerans 1.2244 JF753404 133 18Escherichia coli NBRI1707 JF753405 134 25 Methylobacterium radiotoleransJF753406 135 18 Escherichia coli NBRI1707 JF753407 136 18 Enterobactersp. TSSAS2-21 JF753408 137 18 Enterobacter sp. FMB-1 JF753409 138 18Enterobacter sp. TSSAS2-21 JF753410 139 101 Sphingomonas sp. BF14JF753411 140 18 Hafnia alvei JF753412 141 149 Escherichia coli NBRI1707JF753413 142 27 Burkholderia gladioli pv. Agaricicola JF753414 143 18Escherichia coli NBRI1707 JF753415 144 25 Methylobacterium radiotoleransJF753416 145 194 Micrococcus luteus NBSL29 JF753417 146 37 Burkholderiaphytofirmans PsJN JF753418 147 38 Staphylococcus warneri R-36520JF753419 148 160 Pseudomonas fluorescens JF753420 149 18 Enterobactercloacae C111 JF753421 150 161 Methylobacterium brachiatum JF753422 15127 Burkholderia gladioli pv. agaricicola JF753423 152 18 Escherichiacoli NBRI1707 JF753424 153 16 Staphylococcus sp. SRC_DSF7 JF753425 15467 Staphylococcus epidermitis JF753426 155 64 Methylobacteriumbrachiatum JF753427 156 1 Pseudomonas putida CM5002 JF753428 157 37Burkholderia phytofirmans PsJN JF753429 158 1 Pseudomonas putida CM5002JF753430 159 101 Sphingomonas sp. P5-5 JF753431 160 84 Pantoeaagglomerans CLJ1 JF753432 161 84 Pantoea agglomerans KJPB2 JF753433 1627 Pantoea dispersa CIP 102701 JF753434 163 18 Enterobacter cloacaeR10-1A JF753435 164 1 Pseudomonas putida CM5002 JF753436 165 1Pseudomonas putida CM5002 JF753437 166 0 Pantoea agglomerans KJPB2JF753438 167 143 Pantoea agglomerans KJPB2 JF753439 168 65 Pseudomonasputida CM5002 JF753440 169 1 Pseudomonas tolaasii IExb JF753441 170 84Pantoea agglomerans KJPB2 JF753442 171 2 Pseudomonas oryzihabitansJF753443 172 1 Pseudomonas putida CM5002 JF753444 173 1 Pseudomonasputida CM5002 JF753445 174 143 Pantoea agglomerans KJPB2 JF753446 175164 Pantoea agglomerans KJPB2 JF753447 176 56 Enterobacter asburiae MY2JF753448 177 0 Enterobacter asburiae NFSt10 JF753449 178 25Methylobacterium radiotolerans JF753450 179 7 Pantoea dispersa NCPPB2285 JF753451 180 1 Pseudomonas putida CM5002 JF753452 181 72Cellulomonas denverensis JF753453 182 102 Arthrobacter ramosus JF753454183 72 Cellulomonas denverensis JF753455 184 0 Pantoea ananatis LMG20103 JF753456 185 0 Pantoea ananatis LMG 20103 JF753457 186 102Arthrobacter sp. XY9 JF753458 187 0 Enterobacter asburiae JF753459 188 0Enterobacter cloaceae JF753460 189 196 Enterobacter hormaechei JF753461190 7 Pantoea dispersa NCPPB 2285 JF753462 191 0 Enterobacter cloacae TUJF753463 192 10 Stenotrophomonas maltophilia JF753464 193 10Stenotrophomonas maltophilia JF753465 194 21 Klebsiella pneumoniae 342JF753466 195 0 Citrobacter freundii GM1 JF753467 196 31 Paenibacilluscaespitis JF753468 197 31 Paenibacillus graminis JF753469 198 178Paenibacillus sp. P117 JF753470 199 178 Paenibacillus sp. MK17 JF753471200 72 Cellulomonas denverensis JF753472 201 28 Microbacterium sp.13635I JF753473 202 31 Paenibacillus caespitis JF753474 203 31Paenibacillus caespitis JF753475 204 196 Enterobacter asburiae J2S4JF753476 205 66 Rhizobium sp. HGR13 JF753477 206 84 Pantoea agglomeransKJPB2 JF753478 207 84 Pantoea agglomerans KJPB2 JF753479 208 84 Pantoeaagglomerans Sc-1 JF753480 209 84 Pantoea agglomerans KJPB2 JF753481 2101 Pseudomonas putida CM5002 JF753482 211 2 Pseudomonas sp. TE9 JF753483212 84 Pantoea agglomerans 1.2244 JF753484 213 1 Pseudomonas synxanthaJF753485 214 1 Pseudomonas fluorescens JF753486 215 1 Pseudomonas putidaCM5002 JF753487 216 1 Pseudomonas fluorescens PGPR1 JF753488 217 0Pantoea vagans C9-1 JF753489 218 10 Stenotrophomonas maltophiliaJF753490 219 0 Pantoea agglomerans 1.2244 JF753491 220 37 Burkholderiaphytofirmans PSjN JF753492 221 37 Burkholderia phytofirmans PsJNJF753493 222 92 Streptomyces sp. KN-0260 JF753494 223 64Methylobacterium brachiatum JF753495 224 53 Paenibacillus sp. IHB B 2257JF753496 225 0 Pantoea agglomerans 1.2244 JF753497 226 82 Luteibactersp. MDA0897 JF753498 227 10 Stenotrophomonas maltophilia JF753499 228 7Pantoea dispersa NCPPB 2285 JF753500 229 18 Klebsiella sp. EH47 JF753501230 10 Stenotrophomonas maltophilia JF753502 231 10 Stenotrophomonasmaltophilia JF753503 232 10 Stenotrophomonas maltophilia JF753504 233 10Stenotrophomonas maltophilia JF753505 234 10 Stenotrophomonasmaltophilia JF753506 235 0 Pantoea agglomerans 1.2244 JF753507 236 10Stenotrophomonas maltophilia JF753508 237 192 Enterobacter asburiae MY2JF753509 238 10 Stenotrophomonas maltophilia JF753510 239 22 Bacillusmegaterium NBAII-63 JF753511 240 202 Deinococcus grandis DSM JF753512241 204 Azospirillum zea Gr24 JF753513 242 30 Rhodococcus fasciansNKCM8906 JF753514 243 28 Microbacterium sp. VKM Ac-1389 JF753515 244 41Bacillus subtilis JF753516 245 41 Bacillus subtilis TAT1-8 JF753517 246118 Bacillus asahai NBPP91 JF753518 247 64 Methylobacterium brachiatumJF753519 248 74 Bradyrhizobium japonicum JF753520 249 53 Paenibacillussp. IB-1067 JF753521 250 120 Paenibacillus polymyxa JF753522 251 145Brevibacillus agri JF753523 252 7 Pantoea agglomerans ZFJ-6 JF753524 25356 Enterobacter sp. pp9c JF753525 254 110 Sediminibacterium sp. I-28JF753526 255 200 Bacillus pumilus ustb-06 JF753527 256 39 Bacilluspumilus PhyCEm-115 JF753528 257 76 Bacillus circulans WZ12 JF753529 25876 Bacillus nealsonii PAB1C3 JF753530 259 7 Pantoea agglomerans ZFJ-6JF753531 260 39 Bacillus pumilus CT3 JF753532 261 18 Enterobacter sp.G-2-10-2 JF753533 262 7 Pantoea agglomerans BJTZ1 JF753534 263 120Paenibacillus polymyxa JF753535 264 119 Enterococcus gallinarum JF753536265 0 Enterobacter asburiae M16 JF753537 266 56 Pantoea agglomeransWAB1925 JF753538 267 113 Microbacterium schleiferi JF753539 268 71Sediminibacterium sp. I-28 JF753540 269 119 Enterococcus gallinarumJF753541 270 64 Methylobacterium brachiatum JF753542 271 0 Enterobactercloacae M-5 JF753543 272 39 Bacillus pumilus CT3 JF753544 273 56Enterobacter cloacae JF753545 274 0 Enterobacter cloacae M-5 JF753546275 0 Enterobacter cloacae M-5 JF753547 276 50 Enterobacter hormaecheiskg0061 JF753548 277 170 Pantoea sp. JF753549 278 64 Methylobacteriumbrachiatum JF753550 279 106 Enterobacter asburiae M16 JF753551 280 176Bacillus pumilus NBJ7 JF753552 281 1 Pseudomonas protegens CHA0 JN110435282 10 Stenotrophomonas maltophilia IAM 12423 JN110431 283 10Stenotrophomonas maltophilia IAM 12423 JN110437 284 9 Ochrobactrumtritici SCII 24 JN110432 285 9 Ochrobactrum grignonense OgA9a JN110441286 46 Sphingomonas yanoikuyae IFO 15102 JN110436 287 104 Flavobacteriumjohnsoniae DSM 2064 JN110440 288 24 Paenibacillus humicus PC-147JN110433 289 169 Agromyces mediolanus DSM 20152 JN110439 290 3Curtobacterium citreum DSM 20528 JN110438 291 3 Curtobacterium herbarumDSM 14013 JN110445 292 121 Frigoribacterium faeni DSM 10309 JN110443 293134 Microbacterium oleivorans DSM 16091 JN110444 294 142 Mycobacteriumabscessus CIP 104536 JN110430 295 142 Mycobacterium abscessus CIP 104536JN110434 296 201 Plantibacter flavus DSM 14012 JN110442 297 83Enterobacter cloacae subsp. ATCC 13047 JN110446 cloacae 298 2Pseudomonas oryzihabitans IAM 1568 JN110447 299 193 Aeromonas hydrophilasubsp. LMG 19562 JN110448 dhakensis 300 180 Herbaspirillumrubrisubalvicans ICMP 5777T JN110449 301 23 Acinetobacter beijerinckiiLUH 4759 JN110450 302 66 Rhizobium radiobacter IAM 12048 JN110451 303 18Enterobacter arachidis Ah-143 JN110452 304 83 Escherichia coli O111:Hstr. 11128 JN110453 305 10 Stenotrophomonas maltophilia IAM 12423JN110454 306 84 Pantoea agglomerans DSM3493 JN110455 307 63 Neisseriameningitidis M01-240149 JN110456 308 1 Pseudomonas protegens CHA0JN110457 309 89 Dyella ginsengisoli Gsoil 3046 JN110458 310 2Pseudomonas putida BIRD-1 JN110459 311 19 Bacilluspsychrosaccharolyticus S156 JN110460 312 129 Deinococcus ficus CC-FR2-10JN110461 313 13 Achromobacter spanius LMG 5911 JN110462 314 0 Tatumellamorbirosei JN167639 315 56 Leclercia adecarboxylata JN167641 316 18Enterobacter dissolvens JN167642 317 56 Enterobacter cancerogenusJN167646 318 21 Serratia marcescens JN167643 319 0 Erwinia cypripediJN167644 320 7 Erwinia aphidicola JN167651 321 46 Sphingomonasyanoikuyae JN167645 322 0 Pantoea anthophila JN167647 323 7 Pantoeadispersa JN167640 324 15 Oxalophagus oxalicus JN167648 325 14Paenibacillus nanensis JN167650 326 5 Bosea vestrisii JN167652 327 69Rheinheimera soli JN167653 328 26 Acinetobacter baumannii JN167654 32923 Acinetobacter johnsonii JN167660 330 208 Acinetobacter beijerinckiiJN167680 331 208 Acinetobacter schindleri JN167685 332 116 Roseatelesdepolymerans JN167655 333 116 Roseateles terrae JN167663 334 27Burkholderia diffusa JN167657 335 211 Sphingopyxis panaciterrae JN167658336 98 Massilia aerolata JN167682 337 51 Massilia albidiflava JN167661338 1 Pseudomonas poae JN167662 339 75 Ancylobacter rudongensis JN167664340 10 Stenotrophomonas pavanii JN167665 341 83 Shigella flexneriJN167666 342 91 Bdellovibrio bacteriovorus JN167671 343 56 Enterobactercancerogenus JN167674 344 130 Enhydrobacter aerosaccus JN167675 345 100Variovorax boronicumulans JN167676 346 128 Oceanibaculum pacificumJN167677 347 46 Sphingomonas yanoikuyae JN167683 348 157 Devosiariboflavina JN167684 349 18 Escherichia coli JN167686 350 190Sphingosinicella xenopeptidilytica JN167688 351 120 Paenibacillusdaejeonensis JN167679 352 6 Paenibacillus xylanilyticus JN167687 353 163Sediminibacillus halophilus JN167689 354 44 Corynebacteriumpseudogenitalium JN167659 355 123 Nocardia soli JN167670 356 206 Lentzeaflaviverrucosa JN167672 357 198 Flavobacterium degerlachei JN167656 358165 Flavobacterium aquatile JN167669 359 62 Chryseobacterium hominisJN167678 360 186 Uncultured bacterium Uncultured bacterium JN167667 361195 Uncultured bacterium Uncultured bacterium JN167681 362 21 Klebsiellavariicola JN167690 363 18 Klebsiella pneumoniae JN167691 364 1Pseudomonas plecoglossicida JN167693 365 10 Stenotrophomonas pavaniiJN167694 366 101 Sphingomonas echinoides JN167695 367 66 Rhizobiummassiliae JN167696 368 0 Serratia marcescens JN167697 369 101Sphingomonas echinoides JN167698 370 114 Sphingomonas dokdonensisJN167701 371 7 Pantoea dispersa JN167699 372 82 Luteibacter anthropiJN167700 373 27 Burkholderia gladioli JN167702 374 56 Leclerciaadecarboxylata JN167703 375 167 Tepidimonas aquatic JN167705 376 0Tatumella morbirosei JN167706 377 56 Enterobacter cancerogenus JN167707378 124 Thermomonas brevis JN167708 379 79 Lactobacillus iners JN167704380 7 Pantoea dispersa JN167709 381 101 Sphingomonas echinoides JN167710382 7 Pantoea dispersa JN167784 383 84 Pantoea agglomerans JN167785 384101 Sphingomonas echinoides JN167786 385 101 Sphingomonas echinoidesJN167713 386 18 Shigella flexneri JN167714 387 0 Leclerciaadecarboxylata JN167716 388 29 Pseudoxanthomonas kaohsiungensis JN167717389 57 Psychrobacter pulmonis JN167718 390 100 Variovorax boronicumulansJN167720 391 56 Enterobacter sp. JN167721 392 181 Microvirga aerophilusJN167727 393 132 Microvirga aerilata JN167734 394 7 Erwinia aphidicolaJN167725 395 162 Methylobacterium platani JN167729 396 0 Tatumellamorbirosei JN167730 397 37 Burkholderia phytofirmans JN167732 398 27Burkholderia sp. JN167723 399 36 Acidovorax temperans JN167733 400 0Serratia marcescens JN167743 401 56 Serratia ureilytica JN167737 402 23Acinetobacter beijerinckii JN167738 403 26 Acinetobacter junii JN167739404 23 Acinetobacter johnsonii JN167724 405 23 Acinetobacterkyonggiensis JN167726 406 152 Halomonas daqingensis JN167741 407 7Pantoea dispersa JN167736 408 79 Lactobacillus iners JN167712 409 22Bacillus aryabhattai JN167715 410 67 Staphylococcus hominis JN167722 41167 Staphylococcus capitis JN167728 412 85 Finegoldia magna JN167735 41320 Ruminococcus bromii JN167740 414 42 Aerococcus urinaeequi JN167742415 32 Propioniciclava tarda JN167711 416 70 Propionibacterium acnesJN167719 417 107 Uncultured bacterium JN167731 418 99 Brevundimonasdiminuta JN167744 419 99 Brevundimonas naejangsanensis JN167764 420 101Sphingomonas echinoides JN167745 421 126 Sphingomonas koreensis JN167756422 191 Sphingomonas humi JN167758 423 100 Acidovorax facilis JN167746424 36 Acidovoraz temperans JN167757 425 136 Shinella zoogloeoidesJN167747 426 116 Roseateles depolymerans JN167748 427 116 Roseatelesterrae JN167752 428 69 Rheinheimera chironomi JN167749 429 69Rheinheimera soli JN167775 430 23 Acinetobacter johnsonii JN167750 431208 Acinetobacter schindleri JN167761 432 23 Acinetobacter lwoffiiJN167765 433 2 Pseudomonas stutzeri JN167751 434 184 Thermomonaskoreensis JN167753 435 27 Burkholderia sp. JN167754 436 18 Shigellaflexneri JN167760 437 97 Cellvibrio mixtus JN167766 438 21 Serratiamarcescens JN167767 439 131 Thiobacillus aquaesulis JN167768 440 133Luteimonas aestuarii JN167769 441 197 Sphingosinicella sp. JN167772 442108 Acidithiobacillus albertensis JN167773 443 36 Curvibacter gracilisJN167774 444 47 Devosia insulae JN167777 445 93 Cupriavidus gilardiiJN167778 446 140 Methylobacterium rhodesianum JN167779 447 89 Dokdonellasp. JN167780 448 150 Desemzia incerta JN167763 449 68 Kocuria roseaJN167770 450 123 Nocardia ignorata JN167771 451 182 Pseudonocardiaaurantiaca JN167776 452 104 Flavobacterium johnsoniae JN167755 453 203Flavobacterium mizutaii JN167762 454 73 Flavisolibacter ginsengiterraeJN167781 455 33 Sphingobacterium daejeonense JN167759 456 0 Leclerciaadecarboxylata JN167782 457 56 Enterobacter cancerogenus JN167783 458 39Bacillus altitudinis HQ432811 459 19 Bacillus simplex HQ432812 460 12Bacillus thuringiensis HQ432813 461 6 Paenibacillus amylolyticusHQ432814 462 103 Staphylococcus aureus subsp. aureus HQ432815 463 146Pantoea ananatis AB178169 464 56 Pantoea ananatis AB178170 465 12Bacillus cereus AB178171 466 59 Pantoea ananatis AB178172 467 12Bacillus cereus AB178173 468 45 Sphingomonas echinoides AB178174 469 45Sphingomonas echinoides AB178175 470 45 Sphingomonas echinoides AB178176471 45 Sphingomonas parapaucimobilis AB178177 472 12 Bacillus cereusAB178178 473 12 Bacillus cereus AB178179 474 12 Bacillus cereus AB178192475 12 Bacillus cereus AB178193 476 12 Bacillus cereus AB178194 477 12Bacillus cereus AB178195 478 12 Bacillus cereus AB178196 479 12 Bacilluscereus AB178197 480 12 Bacillus cereus AB178198 481 12 Bacillus cereusAB178199 482 12 Bacillus cereus AB178200 483 12 Bacillus cereus AB178201484 12 Bacillus cereus AB178214 485 12 Bacillus cereus AB178215 486 12Bacillus cereus AB178216 487 12 Bacillus cereus AB178217 488 12 Bacilluscereus AB178218 489 29 Xanthomonas translucens pv. poae AB242936 490 7Pantoea ananatis AB242937 491 7 Pantoea ananatis AB242938 492 8Methylobacterium aquaticum AB242939 493 8 Methylobacterium aquaticumAB242940 494 172 Sphingomonas melonis AB242941 495 45 Sphingomonasyabuuchiae AB242942 496 45 Sphingomonas yabuuchiae AB242943 497 8Methylobacterium aquaticum AB242944 498 7 Pantoea ananatis AB242945 4997 Pantoea ananatis AB242946 500 41 Bacillus subtilis AB242958 501 41Bacillus subtilis AB242959 502 41 Bacillus subtilis AB242960 503 41Bacillus subtilis AB242961 504 39 Bacillus pumilus AB242962 505 59Micrococcus luteus AB242963 506 45 Sphingomonas yabuuchiae AB242964 507148 Sphingomonas yabuuchiae AB242965 508 212 Acidovorax sp. AB242966 5093 Curtobacterium flaccumfaciens pv. Basellae AB242967 510 6Paenibacillus amylolyticus AB242978 511 146 Pantoea ananatis AB242979512 77 Pantoea ananatis AB242980 513 39 Bacillus pumilus AB242981 514 77Pantoea ananatis AB242982 515 29 Xanthomonas translucens AB242983 516 39Bacillus pumilus AB242984 517 3 Curtobacterium flaccumfaciens pv.Basellae AB242985 518 29 Xanthomonas translucens pv. poae AB242986 51939 Bacillus pumilus AB242987 520 29 Xanthomonas translucens pv. poaeAB242988

TABLE 2 Endophytic bacteria isolated from corn, rice and wheat seeds,including assignment to specific OTUs, corresponding Sequence IDnumbers, Family, Genus, Taxonomic information and plant source fromwhich the microbe was derived. SEQ ID Seed-Origin Seed-Origin Source ofseed- Family of Seed- Taxonomy of Seed- Strain OTU# NO: Crop TypeCultivar Type origin microbes Origin Microbe Origin Microbe SYM00033 0541 Teosinte Wild relative Surface sterilized seeds EnterobacteriaceaeEnterobacter sp. SYM00173 0 593 Rice Modern Surface sterilized seedsEnterobacteriaceae Pantoea sp. SYM00176 0 596 Oryza nivara Wild relativeSurface sterilized seeds Enterobacteriaceae Pantoea sp. SYM00284 0 633Maize Modern Surface sterilized seeds Enterobacteriaceae Pantoeaananatis SYM00605 0 716 Maize Modern Seed surface washEnterobacteriaceae SYM00607 0 717 Maize Modern Seed surface washEnterobacteriaceae SYM00608 0 718 Maize Modern Seed surface washEnterobacteriaceae Pantoea sp. SYM00620 0 720 Teosinte Wild relativeSeed surface wash Enterobacteriaceae Enterobacter sp. SYM00658 0 736Avena sterilis Wild relative Seed surface wash EnterobacteriaceaeSYM00985 0 851 Rice Modern Surface sterilized seeds EnterobacteriaceaeSYM01006 0 866 Rice Modern Surface sterilized seeds EnterobacteriaceaeSYM01035 0 887 Avena sterilis Wild relative Surface sterilized seedsEnterobacteriaceae SYM01041 0 892 Rice Ancient Landrace Surfacesterilized seeds Enterobacteriaceae Pantoea sp. SYM01158 0 937 Avenasterilis Wild relative Roots & Seeds Enterobacteriaceae SYM01173 0 943Rice Ancient Landrace Roots & Seeds Enterobacteriaceae SYM01231 0 980Rice Modern Roots & Seeds Enterobacteriaceae SYM00472 1 636 Maize ModernRoots Pseudomonadaceae Pseudomonas sp. SYM00660 1 737 Avena sterilisWild relative Seed surface wash Pseudomonadaceae Pseudomonas sp.SYM00011 2 522 Teosinte Wild relative Surface sterilized seedsPseudomonadaceae Pseudomonas sp. SYM00011b 2 523 Teosinte Wild relativeSurface sterilized seeds Pseudomonadaceae Pseudomonas sp. SYM00013 2 524Teosinte Wild relative Surface sterilized seeds PseudomonadaceaePseudomonas sp. SYM00014 2 526 Teosinte Wild relative Surface sterilizedseeds Pseudomonadaceae Pseudomonas sp. SYM00062 2 557 Teosinte Wildrelative Surface sterilized seeds Pseudomonadaceae Pseudomonas sp.SYM00067 2 562 Teosinte Wild relative Surface sterilized seedsPseudomonadaceae Pseudomonas sp. SYM00068 2 563 Teosinte Wild relativeSurface sterilized seeds Pseudomonadaceae Pseudomonas sp. SYM00069 2 564Teosinte Wild relative Surface sterilized seeds PseudomonadaceaePseudomonas sp. SYM00646 2 730 Rice Modern Seed surface washPseudomonadaceae Pseudomonas sp. SYM00649 2 733 Rice Modern Seed surfacewash Pseudomonadaceae Pseudomonas sp. SYM00650 2 734 Rice Modern Seedsurface wash Pseudomonadaceae Pseudomonas sp. SYM00657 2 735 Avenasterilis Wild relative Seed surface wash Pseudomonadaceae Pseudomonassp. SYM00672 2 738 Oryza latifolia Wild relative Seed surface washPseudomonadaceae Pseudomonas sp. SYM00709 2 747 Rice Modern Seed surfacewash Pseudomonadaceae Pseudomonas sp. SYM00926 2 804 Rice AncientLandrace Surface sterilized seeds Pseudomonadaceae Pseudomonas sp.SYM00927 2 805 Rice Ancient Landrace Surface sterilized seedsPseudomonadaceae Pseudomonas sp. SYM00946 2 821 Rice Modern Surfacesterilized seeds Pseudomonadaceae Pseudomonas sp. SYM00955 2 828 RiceAncient Landrace Surface sterilized seeds Pseudomonadaceae Pseudomonassp. SYM00970 2 839 Rice Ancient Landrace Surface sterilized seedsPseudomonadaceae Pseudomonas sp. SYM00971 2 840 Rice Ancient LandraceSurface sterilized seeds Pseudomonadaceae Pseudomonas sp. SYM00973 2 842Rice Ancient Landrace Surface sterilized seeds PseudomonadaceaePseudomonas sp. SYM00993 2 857 Oryza officinalis Wild relative Surfacesterilized seeds Pseudomonadaceae Pseudomonas sp. SYM01007 2 867 RiceModern Surface sterilized seeds Pseudomonadaceae Pseudomonas sp.SYM01024 2 880 Oryza nivara Wild relative Surface sterilized seedsPseudomonadaceae Pseudomonas sp. SYM01032 2 885 Avena sterilis Wildrelative Surface sterilized seeds Pseudomonadaceae Pseudomonas sp.SYM01036 2 888 Rice Modern Surface sterilized seeds PseudomonadaceaePseudomonas sp. SYM01164 2 940 Rice Ancient Landrace Roots & SeedsPseudomonadaceae Pseudomonas sp. SYM01171 2 942 Rice Ancient LandraceRoots & Seeds Pseudomonadaceae Pseudomonas sp. SYM01177 2 947 RiceAncient Landrace Roots & Seeds Pseudomonadaceae Pseudomonas sp. SYM011782 948 Rice Ancient Landrace Roots & Seeds Pseudomonadaceae Pseudomonassp. SYM01225 2 975 Rice Modern Roots & Seeds PseudomonadaceaePseudomonas sp. SYM01245 2 988 Rice Ancient Landrace Roots & SeedsPseudomonadaceae Pseudomonas sp. SYM01251 2 989 Rice Ancient LandraceRoots & Seeds Pseudomonadaceae Pseudomonas sp. SYM01254 2 990 RiceAncient Landrace Roots & Seeds Pseudomonadaceae Pseudomonas sp.SYM00013b 3 525 Teosinte Wild relative Surface sterilized seedsMicrobacteriaceae Curtobacterium sp. SYM00167 3 588 Rice Modern Surfacesterilized seeds Microbacteriaceae Curtobacterium sp. SYM00171 3 591Rice Modern Surface sterilized seeds Microbacteriaceae Curtobacteriumsp. SYM00174 3 594 Rye Modern Surface sterilized seeds MicrobacteriaceaeCurtobacterium sp. SYM00178 3 598 Rice Ancient Landrace Surfacesterilized seeds Microbacteriaceae Curtobacterium sp. SYM00180 3 600Rice Ancient Landrace Surface sterilized seeds MicrobacteriaceaeCurtobacterium sp. SYM00181 3 601 Rice Ancient Landrace Surfacesterilized seeds Microbacteriaceae Curtobacterium sp. SYM00235 3 622Rice Modern Surface sterilized seeds Microbacteriaceae Curtobacteriumsp. SYM00244 3 626 Barley Modern Surface sterilized seedsMicrobacteriaceae Curtobacterium sp. SYM00525 3 654 Oryza nivara Wildrelative Seed surface wash Microbacteriaceae Curtobacterium sp. SYM006253 724 Maize Modern Seed surface wash Microbacteriaceae Curtobacteriumsp. SYM00645 3 729 Rice Modern Seed surface wash MicrobacteriaceaeCurtobacterium sp. SYM00647 3 731 Rice Modern Seed surface washMicrobacteriaceae Curtobacterium sp. SYM00673b 3 739 Oryza latifoliaWild relative Seed surface wash Microbacteriaceae Curtobacterium sp.SYM00690 3 740 Rice Modern Seed surface wash MicrobacteriaceaeCurtobacterium sp. SYM00691 3 741 Rice Modern Seed surface washMicrobacteriaceae Curtobacterium sp. SYM00693 3 742 Rice Modern Seedsurface wash Microbacteriaceae Curtobacterium sp. SYM00694b 3 744 RiceModern Seed surface wash Microbacteriaceae Curtobacterium sp. SYM00712 3748 Rice Modern Seed surface wash Microbacteriaceae Curtobacterium sp.SYM00716 3 752 Rice Ancient Landrace Seed surface wash MicrobacteriaceaeCurtobacterium sp. SYM00722 3 753 Rice Ancient Landrace Seed surfacewash Microbacteriaceae Curtobacterium sp. SYM00722B 3 754 Rice AncientLandrace Seed surface wash Microbacteriaceae Curtobacterium sp.SYM00731B 3 756 Rice Ancient Landrace Seed surface washMicrobacteriaceae Curtobacterium sp. SYM00749 3 758 Rice AncientLandrace Surface sterilized seeds Microbacteriaceae Curtobacterium sp.SYM00784 3 773 Maize Modern Seed surface wash MicrobacteriaceaeCurtobacterium sp. SYM00947 3 822 Rice Modern Surface sterilized seedsMicrobacteriaceae Curtobacterium sp. SYM00949 3 823 Rice Modern Surfacesterilized seeds Microbacteriaceae Curtobacterium sp. SYM00952 3 826Rice Ancient Landrace Surface sterilized seeds MicrobacteriaceaeCurtobacterium sp. SYM00964 3 834 Rice Ancient Landrace Surfacesterilized seeds Microbacteriaceae Curtobacterium sp. SYM00976 3 844Rice Ancient Landrace Surface sterilized seeds MicrobacteriaceaeCurtobacterium sp. SYM00980 3 847 Rice Modern Surface sterilized seedsMicrobacteriaceae Curtobacterium sp. SYM00984 3 850 Rice Modern Surfacesterilized seeds Microbacteriaceae Curtobacterium sp. SYM00996 3 859Oryza officinalis Wild relative Surface sterilized seedsMicrobacteriaceae Curtobacterium sp. SYM01013 3 872 Rice AncientLandrace Surface sterilized seeds Microbacteriaceae Curtobacterium sp.SYM01022 3 879 Oryza nivara Wild relative Surface sterilized seedsMicrobacteriaceae Curtobacterium sp. SYM01025 3 881 Oryza nivara Wildrelative Surface sterilized seeds Microbacteriaceae Curtobacterium sp.SYM01142 3 928 Rice Modern Roots & Seeds MicrobacteriaceaeCurtobacterium sp. SYM01144 3 929 Rice Modern Roots & SeedsMicrobacteriaceae Curtobacterium sp. SYM01148 3 931 Rice Modern Roots &Seeds Microbacteriaceae Curtobacterium sp. SYM01151 3 932 Rice ModernRoots & Seeds Microbacteriaceae Curtobacterium sp. SYM01155 3 935 RiceModern Roots & Seeds Microbacteriaceae Curtobacterium sp. SYM01156 3 936Rice Modern Roots & Seeds Microbacteriaceae Curtobacterium sp. SYM011793 949 Rice Modern Roots & Seeds Microbacteriaceae Curtobacterium sp.SYM01181 3 951 Rice Modern Roots & Seeds MicrobacteriaceaeCurtobacterium sp. SYM01182 3 952 Rice Modern Roots & SeedsMicrobacteriaceae Curtobacterium sp. SYM01183 3 953 Rice Modern Roots &Seeds Microbacteriaceae Curtobacterium sp. SYM01184 3 954 Rice ModernRoots & Seeds Microbacteriaceae Curtobacterium sp. SYM01185 3 955 RiceModern Roots & Seeds Microbacteriaceae Curtobacterium sp. SYM01188 3 957Rice Modern Roots & Seeds Microbacteriaceae Curtobacterium sp. SYM011983 962 Rice Modern Roots & Seeds Microbacteriaceae Curtobacterium sp.SYM01199 3 963 Rice Modern Roots & Seeds MicrobacteriaceaeCurtobacterium sp. SYM01201 3 964 Rice Modern Roots & SeedsMicrobacteriaceae Curtobacterium sp. SYM01202 3 965 Rice Modern Roots &Seeds Microbacteriaceae Curtobacterium sp. SYM01204 3 966 Rice ModernRoots & Seeds Microbacteriaceae Curtobacterium sp. SYM01205 3 967 RiceModern Roots & Seeds Microbacteriaceae Curtobacterium sp. SYM01207 3 969Rice Modern Roots & Seeds Microbacteriaceae Curtobacterium sp. SYM012153 971 Rice Modern Roots & Seeds Microbacteriaceae Curtobacterium sp.SYM01218 3 973 Rice Modern Roots & Seeds MicrobacteriaceaeCurtobacterium sp. SYM01222 3 974 Rice Modern Roots & SeedsMicrobacteriaceae Curtobacterium sp. SYM00188 6 605 Maize Modern LeavesPaenibacillaceae Paenibacillus sp. SYM00190 6 607 Maize Modern LeavesPaenibacillaceae Paenibacillus sp. SYM00195 6 610 Maize Modern LeavesPaenibacillaceae Paenibacillus sp. SYM00217 6 616 Soybean Modern RootsPaenibacillaceae Paenibacillus sp. SYM00227 6 619 Soybean Modern LeavesPaenibacillaceae Paenibacillus sp. SYM00292 6 634 Maize Modern Surfacesterilized seeds Paenibacillaceae Paenibacillus taichungensis SYM00597 6711 Maize Ancient Landrace Seed surface wash PaenibacillaceaePaenibacillus sp. SYM01108 6 915 Oryza nivara Wild relative Surfacesterilized seeds Paenibacillaceae Paenibacillus sp. SYM01109 6 916 Oryzanivara Wild relative Surface sterilized seeds PaenibacillaceaePaenibacillus sp. SYM01110 6 917 Oryza nivara Wild relative Surfacesterilized seeds Paenibacillaceae Paenibacillus sp. SYM01111 6 918 Oryzanivara Wild relative Surface sterilized seeds PaenibacillaceaePaenibacillus sp. SYM01112 6 919 Oryza nivara Wild relative Surfacesterilized seeds Paenibacillaceae Paenibacillus sp. SYM01114 6 921 MaizeModern Roots Paenibacillaceae Paenibacillus sp. SYM01117 6 922 MaizeAncient Landrace Roots Paenibacillaceae Paenibacillus sp. SYM01118 6 923Maize Ancient Landrace Roots Paenibacillaceae Paenibacillus sp. SYM011276 925 Teosinte Wild relative Roots Paenibacillaceae Paenibacillus sp.SYM01256 6 991 Maize Ancient Landrace Roots PaenibacillaceaePaenibacillus sp. SYM00014b 7 527 Teosinte Wild relative Surfacesterilized seeds Enterobacteriaceae Erwinia sp. SYM00017b 7 532 RiceModern Surface sterilized seeds Enterobacteriaceae Pantoea sp. SYM000187 534 Maize Ancient Landrace Surface sterilized seeds EnterobacteriaceaePantoea sp. SYM00020 7 535 Maize Ancient Landrace Surface sterilizedseeds Enterobacteriaceae Pantoea sp. SYM00022 7 537 Teosinte Wildrelative Surface sterilized seeds Enterobacteriaceae Pantoea sp.SYM00025 7 538 Maize Ancient Landrace Surface sterilized seedsEnterobacteriaceae Pantoea sp. SYM00026 7 539 Maize Ancient LandraceSurface sterilized seeds Enterobacteriaceae Pantoea sp. SYM00043 7 544Maize Modern Surface sterilized seeds Enterobacteriaceae Pantoea sp.SYM00047 7 546 Maize Ancient Landrace Surface sterilized seedsEnterobacteriaceae Pantoea sp. SYM00049 7 547 Maize Ancient LandraceSurface sterilized seeds Enterobacteriaceae Pantoea sp. SYM00055 7 553Maize Ancient Landrace Surface sterilized seeds EnterobacteriaceaePantoea sp. SYM00057 7 554 Maize Ancient Landrace Surface sterilizedseeds Enterobacteriaceae Pantoea sp. SYM00058 7 555 Maize AncientLandrace Surface sterilized seeds Enterobacteriaceae Pantoea sp.SYM00078 7 568 Maize Ancient Landrace Surface sterilized seedsEnterobacteriaceae Pantoea sp. SYM00081 7 569 Maize Ancient LandraceSeed surface wash Enterobacteriaceae Pantoea sp. SYM00082a 7 570 MaizeAncient Landrace Seed surface wash Enterobacteriaceae Pantoea sp.SYM00085 7 571 Maize Modern Surface sterilized seeds EnterobacteriaceaePantoea sp. SYM00086 7 572 Maize Modern Surface sterilized seedsEnterobacteriaceae Pantoea sp. SYM00087 7 573 Maize Maize PI 485356Surface sterilized seeds Enterobacteriaceae Pantoea sp. SYM00088 7 574Maize Maize PI 485356 Surface sterilized seeds EnterobacteriaceaePantoea sp. SYM00094 7 576 Maize Ancient Landrace Surface sterilizedseeds Enterobacteriaceae Pantoea sp. SYM00095 7 577 Maize AncientLandrace Surface sterilized seeds Enterobacteriaceae Pantoea sp.SYM00096 7 578 Maize Ancient Landrace Surface sterilized seedsEnterobacteriaceae Pantoea sp. SYM00100 7 579 Maize Ancient LandraceSurface sterilized seeds Enterobacteriaceae Pantoea sp. SYM00101 7 580Maize Ancient Landrace Surface sterilized seeds EnterobacteriaceaePantoea sp. SYM00502 7 639 Maize Ancient Landrace Seed surface washEnterobacteriaceae Erwinia sp. SYM00506 7 641 Maize Ancient LandraceSeed surface wash Enterobacteriaceae Erwinia sp. SYM00506b 7 642 MaizeAncient Landrace Seed surface wash Enterobacteriaceae Erwinia sp.SYM00511 7 647 Maize Ancient Landrace Seed surface washEnterobacteriaceae Erwinia sp. SYM00514b 7 649 Maize Ancient LandraceSeed surface wash Enterobacteriaceae Erwinia sp. SYM00514C 7 650 MaizeAncient Landrace Seed surface wash Enterobacteriaceae Erwinia sp.SYM00514D 7 651 Maize Ancient Landrace Seed surface washEnterobacteriaceae Erwinia sp. SYM00731A 7 755 Rice Ancient LandraceSeed surface wash Enterobacteriaceae Erwinia sp. SYM00785 7 774 MaizeModern Seed surface wash Enterobacteriaceae Erwinia sp. SYM01056 7 903Teosinte Wild relative Surface sterilized seeds EnterobacteriaceaeErwinia sp. SYM01235 7 984 Oryza officinalis Wild relative Roots & SeedsEnterobacteriaceae Erwinia sp. SYM01238 7 986 Oryza officinalis Wildrelative Roots & Seeds Enterobacteriaceae Erwinia sp. SYM00967 8 837Rice Ancient Landrace Surface sterilized seeds MethylobacteriaceaeSYM01233 8 982 Oryza officinalis Wild relative Roots & SeedsMethylobacteriaceae SYM00544 9 663 Maize Ancient Landrace Seed surfacewash Brucellaceae Ochrobactrum sp. SYM00545B 9 665 Maize AncientLandrace Seed surface wash Brucellaceae Ochrobactrum sp. SYM00548 9 667Maize Ancient Landrace Seed surface wash Brucellaceae Ochrobactrum sp.SYM00552 9 670 Maize Ancient Landrace Seed surface wash BrucellaceaeOchrobactrum sp. SYM00558 9 675 Maize Ancient Landrace Seed surface washBrucellaceae Ochrobactrum sp. SYM00580A 9 688 Maize Modern Seed surfacewash Brucellaceae Ochrobactrum sp. SYM00580b 9 689 Maize Modern Seedsurface wash Brucellaceae Ochrobactrum sp. SYM00580d 9 691 Maize ModernSeed surface wash Brucellaceae Ochrobactrum sp. SYM00581d 9 698 MaizeModern Seed surface wash Brucellaceae Ochrobactrum sp. SYM00583 9 699Maize Ancient Landrace Seed surface wash Brucellaceae Ochrobactrum sp.SYM00584 9 700 Maize Ancient Landrace Seed surface wash BrucellaceaeOchrobactrum sp. SYM00588 9 705 Maize Ancient Landrace Seed surface washBrucellaceae Ochrobactrum sp. SYM00596 9 710 Maize Ancient Landrace Seedsurface wash Brucellaceae Ochrobactrum sp. SYM00600 9 713 Maize AncientLandrace Seed surface wash Brucellaceae Ochrobactrum sp. SYM00746 9 757Rice Ancient Landrace Surface sterilized seeds Brucellaceae Ochrobactrumsp. SYM00752 9 759 Maize Modern Seed surface wash BrucellaceaeOchrobactrum sp. SYM00756 9 761 Maize Modern Seed surface washBrucellaceae Ochrobactrum sp. SYM00763 9 767 Maize Modern Seed surfacewash Brucellaceae Ochrobactrum sp. SYM00783 9 772 Maize Modern Seedsurface wash Brucellaceae Ochrobactrum sp. SYM00812 9 775 Rice ModernSeed surface wash Brucellaceae Ochrobactrum sp. SYM00902 9 783 MaizeAncient Landrace Surface sterilized seeds Brucellaceae Ochrobactrum sp.SYM00923 9 802 Maize Modern Surface sterilized seeds BrucellaceaeOchrobactrum sp. SYM00935 9 810 Rice Modern Surface sterilized seedsBrucellaceae Ochrobactrum sp. SYM00937 9 812 Rice Modern Surfacesterilized seeds Brucellaceae Ochrobactrum sp. SYM00954 9 827 RiceAncient Landrace Surface sterilized seeds Brucellaceae Ochrobactrum sp.SYM01029 9 883 Avena sterilis Wild relative Surface sterilized seedsBrucellaceae Ochrobactrum sp. SYM01043 9 894 Rice Modern Surfacesterilized seeds Brucellaceae Ochrobactrum sp. SYM01047 9 896 Oryzalatifolia Wild relative Surface sterilized seeds BrucellaceaeOchrobactrum sp. SYM01052 9 899 Maize Ancient Landrace Surfacesterilized seeds Brucellaceae Ochrobactrum sp. SYM01054 9 901 MaizeAncient Landrace Surface sterilized seeds Brucellaceae Ochrobactrum sp.SYM01055 9 902 Maize Ancient Landrace Surface sterilized seedsBrucellaceae Ochrobactrum sp. SYM01058 9 904 Maize Ancient LandraceSurface sterilized seeds Brucellaceae Ochrobactrum sp. SYM01064 9 906Maize Ancient Landrace Surface sterilized seeds BrucellaceaeOchrobactrum sp. SYM01066 9 908 Maize Ancient Landrace Surfacesterilized seeds Brucellaceae Ochrobactrum sp. SYM01069 9 909 MaizeModern Surface sterilized seeds Brucellaceae Ochrobactrum sp. SYM01079 9913 Maize Modern Surface sterilized seeds Brucellaceae Ochrobactrum sp.SYM00064a 10 560 Teosinte Wild relative Surface sterilized seedsXanthomonadaceae Stenotrophomonas sp. SYM00183 10 603 Oryza glumipatulaWild relative Surface sterilized seeds Xanthomonadaceae Stenotrophomonassp. SYM00184 10 604 Oryza glumipatula Wild relative Surface sterilizedseeds Xanthomonadaceae Stenotrophomonas sp. SYM00905 10 786 Maize ModernSurface sterilized seeds Xanthomonadaceae Stenotrophomonas sp. SYM0054312 662 Maize Ancient Landrace Seed surface wash Bacillaceae Bacillus sp.SYM00595 12 709 Maize Ancient Landrace Seed surface wash BacillaceaeBacillus sp. SYM01227 12 977 Rice Modern Roots & Seeds BacillaceaeBacillus sp. SYM00547 13 666 Maize Ancient Landrace Seed surface washAlcaligenaceae Achromobacter sp. SYM00551 13 669 Maize Ancient LandraceSeed surface wash Alcaligenaceae Achromobacter sp. SYM00560 13 676 MaizeAncient Landrace Seed surface wash Alcaligenaceae Achromobacter sp.SYM00565B 13 681 Maize Modern Seed surface wash AlcaligenaceaeAchromobacter sp. SYM00580C 13 690 Maize Modern Seed surface washAlcaligenaceae Achromobacter sp. SYM00580i 13 694 Maize Modern Seedsurface wash Alcaligenaceae Achromobacter sp. SYM00585 13 701 MaizeAncient Landrace Seed surface wash Alcaligenaceae Achromobacter sp.SYM00586b 13 702 Maize Ancient Landrace Seed surface wash AlcaligenaceaeAchromobacter sp. SYM00588b 13 706 Maize Ancient Landrace Seed surfacewash Alcaligenaceae Achromobacter sp. SYM00591 13 708 Maize AncientLandrace Seed surface wash Alcaligenaceae Achromobacter sp. SYM00602 13715 Maize Modern Seed surface wash Alcaligenaceae Achromobacter sp.SYM00758 13 763 Maize Modern Seed surface wash AlcaligenaceaeAchromobacter sp. SYM00761 13 765 Maize Modern Seed surface washAlcaligenaceae Achromobacter sp. SYM00764 13 768 Maize Modern Seedsurface wash Alcaligenaceae Achromobacter sp. SYM00765 13 769 MaizeModern Seed surface wash Alcaligenaceae Achromobacter sp. SYM00824 13777 Rice Ancient Landrace Seed surface wash Alcaligenaceae Achromobactersp. SYM00828 13 778 Rice Ancient Landrace Seed surface washAlcaligenaceae Achromobacter sp. SYM00830 13 779 Rice Ancient LandraceSeed surface wash Alcaligenaceae Achromobacter sp. SYM00831 13 780 RiceAncient Landrace Seed surface wash Alcaligenaceae Achromobacter sp.SYM00901 13 782 Maize Ancient Landrace Surface sterilized seedsAlcaligenaceae Achromobacter sp. SYM00903 13 784 Maize Modern Surfacesterilized seeds Alcaligenaceae Achromobacter sp. SYM00904 13 785 MaizeModern Surface sterilized seeds Alcaligenaceae Achromobacter sp.SYM00907 13 787 Maize Modern Surface sterilized seeds AlcaligenaceaeAchromobacter sp. SYM00908 13 788 Maize Ancient Landrace Surfacesterilized seeds Alcaligenaceae Achromobacter sp. SYM00909 13 789 MaizeAncient Landrace Surface sterilized seeds Alcaligenaceae Achromobactersp. SYM00910 13 790 Maize Modern Surface sterilized seeds AlcaligenaceaeAchromobacter sp. SYM00914 13 794 Maize Modern Surface sterilized seedsAlcaligenaceae Achromobacter sp. SYM00917 13 796 Maize Modern Surfacesterilized seeds Alcaligenaceae Achromobacter sp. SYM00929 13 806 Oryzalatifolia Wild relative Surface sterilized seeds AlcaligenaceaeAchromobacter sp. SYM00930 13 807 Rice Modern Surface sterilized seedsAlcaligenaceae Achromobacter sp. SYM00938 13 813 Rice Modern Surfacesterilized seeds Alcaligenaceae Achromobacter sp. SYM00957 13 829 RiceAncient Landrace Surface sterilized seeds Alcaligenaceae Achromobactersp. SYM00959 13 830 Rice Ancient Landrace Surface sterilized seedsAlcaligenaceae Achromobacter sp. SYM01017 13 875 Rice Modern Surfacesterilized seeds Alcaligenaceae Achromobacter sp. SYM01020 13 877 RiceModern Surface sterilized seeds Alcaligenaceae Achromobacter sp.SYM01021 13 878 Oryza nivara Wild relative Surface sterilized seedsAlcaligenaceae Achromobacter sp. SYM01030 13 884 Avena sterilis Wildrelative Surface sterilized seeds Alcaligenaceae Achromobacter sp.SYM00028 18 540 Maize Ancient Landrace Surface sterilized seedsEnterobacteriaceae Enterobacter sp. SYM00052 18 550 Teosinte Wildrelative Surface sterilized seeds Enterobacteriaceae Enterobacter sp.SYM00053 18 551 Teosinte Wild relative Surface sterilized seedsEnterobacteriaceae Enterobacter sp. SYM00054 18 552 Teosinte Wildrelative Surface sterilized seeds Enterobacteriaceae Enterobacter sp.SYM00175 18 595 Winter rye Modern Surface sterilized seedsEnterobacteriaceae Enterobacter sp. SYM00627 18 725 Maize Modern Seedsurface wash Enterobacteriaceae Enterobacter sp. SYM00715 18 751 RiceModern Seed surface wash Enterobacteriaceae Enterobacter sp. SYM00189 19606 Maize Modern Leaves Bacillaceae Bacillus sp. SYM00192 19 608 MaizeModern Leaves Bacillaceae Bacillus sp. SYM00197 19 611 Maize ModernLeaves Bacillaceae Bacillus sp. SYM00201 19 612 Maize Maize RootsBacillaceae Bacillus sp. SYM00202 19 613 Maize Maize Roots BacillaceaeBacillus sp. SYM00215 19 615 Soybean Modern Roots Bacillaceae Bacillussp. SYM00233 19 621 Soybean Modern Leaves Bacillaceae Bacillus sp.SYM00260 19 632 Maize Modern Surface sterilized seeds BacillaceaeBacillus simplex SYM01113 19 920 Maize Modern Roots Bacillaceae Bacillussp. SYM01119 19 924 Maize Ancient Landrace Roots Bacillaceae Bacillussp. SYM00016b 25 529 Rice Modern Surface sterilized seedsMethylobacteriaceae Methylobacterium sp. SYM00236 25 623 Rice ModernSurface sterilized seeds Methylobacteriaceae Methylobacterium sp.SYM00237 25 624 Rice Modern Surface sterilized seeds MethylobacteriaceaeMethylobacterium sp. SYM00240 25 625 Rice Modern Surface sterilizedseeds Methylobacteriaceae Methylobacterium sp. SYM00924 25 803 RiceAncient Landrace Surface sterilized seeds MethylobacteriaceaeMethylobacterium sp. SYM00936 25 811 Rice Modern Surface sterilizedseeds Methylobacteriaceae Methylobacterium sp. SYM00950 25 824 RiceAncient Landrace Surface sterilized seeds MethylobacteriaceaeMethylobacterium sp. SYM00968 25 838 Rice Ancient Landrace Surfacesterilized seeds Methylobacteriaceae Methylobacterium sp. SYM00986 25852 Rice Modern Surface sterilized seeds MethylobacteriaceaeMethylobacterium sp. SYM00998 25 861 Oryza officinalis Wild relativeSurface sterilized seeds Methylobacteriaceae Methylobacterium sp.SYM00999 25 862 Oryza officinalis Wild relative Surface sterilized seedsMethylobacteriaceae Methylobacterium sp. SYM01003 25 864 Rice ModernSurface sterilized seeds Methylobacteriaceae Methylobacterium sp.SYM01008 25 868 Rice Modern Surface sterilized seeds MethylobacteriaceaeMethylobacterium sp. SYM00501 27 638 Maize Ancient Landrace Seed surfacewash Burkholderiaceae Burkholderia sp. SYM00504 27 640 Maize AncientLandrace Seed surface wash Burkholderiaceae Burkholderia sp. SYM00536 27656 Maize Ancient Landrace Seed surface wash BurkholderiaceaeBurkholderia sp. SYM00536A 27 657 Maize Ancient Landrace Seed surfacewash Burkholderiaceae Burkholderia sp. SYM00538E 27 659 Maize AncientLandrace Seed surface wash Burkholderiaceae Burkholderia sp. SYM00566A27 682 Maize Modern Seed surface wash Burkholderiaceae Burkholderia sp.SYM00568 27 683 Maize Modern Seed surface wash BurkholderiaceaeBurkholderia sp. SYM00570 27 684 Maize Modern Seed surface washBurkholderiaceae Burkholderia sp. SYM00574 27 685 Maize Ancient LandraceSeed surface wash Burkholderiaceae Burkholderia sp. SYM00575 27 686Maize Ancient Landrace Seed surface wash Burkholderiaceae Burkholderiasp. SYM00578 27 687 Maize Modern Seed surface wash BurkholderiaceaeBurkholderia sp. SYM00621 27 721 Maize Modern Seed surface washBurkholderiaceae Burkholderia sp. SYM00623 27 722 Maize Modern Seedsurface wash Burkholderiaceae Burkholderia sp. SYM00624 27 723 MaizeModern Seed surface wash Burkholderiaceae Burkholderia sp. SYM00633 27727 Maize Ancient Landrace Seed surface wash BurkholderiaceaeBurkholderia sp. SYM00822 27 776 Rice Modern Seed surface washBurkholderiaceae Burkholderia sp. SYM01010 27 869 Rice Ancient LandraceSurface sterilized seeds Burkholderiaceae Burkholderia sp. SYM01012 27871 Rice Ancient Landrace Surface sterilized seeds BurkholderiaceaeBurkholderia sp. SYM01015 27 873 Rice Ancient Landrace Surfacesterilized seeds Burkholderiaceae Burkholderia sp. SYM01037 27 889 RiceModern Surface sterilized seeds Burkholderiaceae Burkholderia sp.SYM00037 28 543 Maize Modern Surface sterilized seeds MicrobacteriaceaeBacillus sp. SYM00051 28 549 Teosinte Wild relative Surface sterilizedseeds Microbacteriaceae Microbacterium sp. SYM00104 28 582 Maize AncientLandrace Surface sterilized seeds Microbacteriaceae Microbacterium sp.SYM00177 28 597 Oryza nivara Wild relative Surface sterilized seedsMicrobacteriaceae Microbacterium sp. SYM00514A 28 648 Maize AncientLandrace Seed surface wash Microbacteriaceae Microbacterium sp. SYM0052328 652 Oryza nivara Wild relative Seed surface wash MicrobacteriaceaeMicrobacterium sp. SYM00538H 28 660 Maize Ancient Landrace Seed surfacewash Microbacteriaceae Microbacterium sp. SYM00542 28 661 Maize AncientLandrace Seed surface wash Microbacteriaceae Microbacterium sp. SYM0055628 674 Maize Ancient Landrace Seed surface wash MicrobacteriaceaeMicrobacterium sp. SYM00581A 28 695 Maize Modern Seed surface washMicrobacteriaceae Microbacterium sp. SYM00586c 28 703 Maize AncientLandrace Seed surface wash Microbacteriaceae Microbacterium sp. SYM0058728 704 Maize Ancient Landrace Seed surface wash MicrobacteriaceaeMicrobacterium sp. SYM00598 28 712 Maize Ancient Landrace Seed surfacewash Microbacteriaceae Microbacterium sp. SYM00757 28 762 Maize ModernSeed surface wash Microbacteriaceae Microbacterium sp. SYM00760 28 764Maize Modern Seed surface wash Microbacteriaceae Microbacterium sp.SYM00780 28 771 Maize Modern Seed surface wash MicrobacteriaceaeMicrobacterium sp. SYM00832 28 781 Rice Ancient Landrace Seed surfacewash Microbacteriaceae Microbacterium sp. SYM00911 28 791 Maize ModernSurface sterilized seeds Microbacteriaceae Microbacterium sp. SYM0091228 792 Maize Ancient Landrace Surface sterilized seeds MicrobacteriaceaeMicrobacterium sp. SYM00913 28 793 Maize Ancient Landrace Surfacesterilized seeds Microbacteriaceae Microbacterium sp. SYM00915 28 795Maize Modern Surface sterilized seeds Microbacteriaceae Microbacteriumsp. SYM00918 28 797 Maize Ancient Landrace Surface sterilized seedsMicrobacteriaceae Microbacterium sp. SYM00919 28 798 Maize AncientLandrace Surface sterilized seeds Microbacteriaceae Microbacterium sp.SYM00920 28 799 Maize Ancient Landrace Surface sterilized seedsMicrobacteriaceae Microbacterium sp. SYM00921 28 800 Maize ModernSurface sterilized seeds Microbacteriaceae Microbacterium sp. SYM0092228 801 Maize Modern Surface sterilized seeds MicrobacteriaceaeMicrobacterium sp. SYM00931 28 808 Rice Modern Surface sterilized seedsMicrobacteriaceae Microbacterium sp. SYM00933 28 809 Rice Modern Surfacesterilized seeds Microbacteriaceae Microbacterium sp. SYM00939 28 814Rice Modern Surface sterilized seeds Microbacteriaceae Microbacteriumsp. SYM00944 28 819 Rice Modern Surface sterilized seedsMicrobacteriaceae Microbacterium sp. SYM00962 28 832 Rice AncientLandrace Surface sterilized seeds Microbacteriaceae Microbacterium sp.SYM01000 28 863 Oryza officinalis Wild relative Surface sterilized seedsMicrobacteriaceae Microbacterium sp. SYM01034 28 886 Avena sterilis Wildrelative Surface sterilized seeds Microbacteriaceae Microbacterium sp.SYM01206 28 968 Rice Modern Roots & Seeds MicrobacteriaceaeMicrobacterium sp. SYM00015 29 528 Rice Modern Surface sterilized seedsXanthomonadaceae Xanthomonas sp. SYM00021 29 536 Teosinte Wild relativeSurface sterilized seeds Xanthomonadaceae Xanthomonas sp. SYM00179 29599 Rice Ancient Landrace Surface sterilized seeds XanthomonadaceaeXanthomonas sp. SYM00182 29 602 Rice Ancient Landrace Surface sterilizedseeds Xanthomonadaceae Xanthomonas sp. SYM00252 29 630 Rice AncientLandrace Surface sterilized seeds Xanthomonadaceae Xanthomonas sp.SYM00977 29 845 Rice Ancient Landrace Surface sterilized seedsXanthomonadaceae Xanthomonas sp. SYM00988 29 854 Rice Modern Surfacesterilized seeds Xanthomonadaceae Xanthomonas sp. SYM00997 29 860 Oryzaofficinalis Wild relative Surface sterilized seeds XanthomonadaceaeXanthomonas sp. SYM01018 29 876 Rice Modern Surface sterilized seedsXanthomonadaceae Xanthomonas sp. SYM01028 29 882 Oryza nivara Wildrelative Surface sterilized seeds Xanthomonadaceae Xanthomonas sp.SYM01146 29 930 Rice Modern Roots & Seeds Xanthomonadaceae Xanthomonassp. SYM01153 29 933 Rice Modern Roots & Seeds XanthomonadaceaeXanthomonas sp. SYM01154 29 934 Rice Modern Roots & SeedsXanthomonadaceae Xanthomonas sp. SYM01162 29 939 Rice Ancient LandraceRoots & Seeds Xanthomonadaceae Xanthomonas sp. SYM01190 29 959 RiceModern Roots & Seeds Xanthomonadaceae Xanthomonas sp. SYM00565A 30 680Maize Modern Seed surface wash Nocardiaceae Rhodococcus sp. SYM00580G 30693 Maize Modern Seed surface wash Nocardiaceae Rhodococcus sp. SYM0075330 760 Maize Modern Seed surface wash Nocardiaceae Rhodococcus sp.SYM00762 30 766 Maize Modern Seed surface wash Nocardiaceae Rhodococcussp. SYM00775 30 770 Maize Modern Seed surface wash NocardiaceaeRhodococcus sp. SYM00943 30 818 Rice Modern Surface sterilized seedsNocardiaceae Rhodococcus sp. SYM00951 30 825 Rice Ancient LandraceSurface sterilized seeds Nocardiaceae Rhodococcus sp. SYM01039 30 890Rice Ancient Landrace Surface sterilized seeds Nocardiaceae Rhodococcussp. SYM01040 30 891 Rice Ancient Landrace Surface sterilized seedsNocardiaceae Rhodococcus sp. SYM01042 30 893 Rice Modern Surfacesterilized seeds Nocardiaceae Rhodococcus sp. SYM01046 30 895 RiceModern Surface sterilized seeds Nocardiaceae Rhodococcus sp. SYM01048 30897 Oryza latifolia Wild relative Surface sterilized seeds NocardiaceaeRhodococcus sp. SYM01053 30 900 Maize Modern Surface sterilized seedsNocardiaceae Rhodococcus sp. SYM01063 30 905 Maize Modern Surfacesterilized seeds Nocardiaceae Rhodococcus sp. SYM01065 30 907 MaizeAncient Landrace Surface sterilized seeds Nocardiaceae Rhodococcus sp.SYM01070 30 910 Rice Modern Surface sterilized seeds NocardiaceaeRhodococcus sp. SYM01071 30 911 Maize Ancient Landrace Surfacesterilized seeds Nocardiaceae Rhodococcus sp. SYM01078 30 912 RiceModern Surface sterilized seeds Nocardiaceae Rhodococcus sp. SYM00589 31707 Maize Ancient Landrace Seed surface wash PaenibacillaceaePaenibacillus sp. SYM00991 36 855 Rice Modern Surface sterilized seedsComamonadaceae Acidovorax sp. SYM01236 36 985 Oryza officinalis Wildrelative Roots & Seeds Comamonadaceae Acidovorax sp. SYM00057B 37 1446Maize Ancient Landrace Surface sterilized seeds BurkholderiaceaeBurkholderia phytofirmans SYM00102 38 581 Maize Ancient Landrace Surfacesterilized seeds Staphylococcaceae Staphylococcus sp. SYM00072 39 566Teosinte Wild relative Surface sterilized seeds Bacillaceae Bacillus sp.SYM00075 39 567 Teosinte Wild relative Surface sterilized seedsBacillaceae Bacillus sp. SYM00249 39 628 Soybean Modern Surfacesterilized seeds Bacillaceae Bacillus sp. SYM00507 39 645 Maize AncientLandrace Seed surface wash Bacillaceae Bacillus sp. SYM00553 39 671Maize Ancient Landrace Seed surface wash Bacillaceae Bacillus sp.SYM00562 39 677 Maize Ancient Landrace Seed surface wash BacillaceaeBacillus sp. SYM00564 39 679 Maize Ancient Landrace Seed surface washBacillaceae Bacillus sp. SYM00580E 39 692 Maize Modern Seed surface washBacillaceae Bacillus sp. SYM00581b 39 696 Maize Modern Seed surface washBacillaceae Bacillus sp. SYM00581c 39 697 Maize Modern Seed surface washBacillaceae Bacillus sp. SYM00601 39 714 Maize Ancient Landrace Seedsurface wash Bacillaceae Bacillus sp. SYM00036 41 542 Maize ModernSurface sterilized seeds Bacillaceae Bacillus sp. SYM00110 41 586 MaizeModern Surface sterilized seeds Bacillaceae Bacillus sp. SYM00193 41 609Maize Modern Leaves Bacillaceae Bacillus sp. SYM00218 41 617 SoybeanModern Roots Bacillaceae Bacillus sp. SYM00250 41 629 Soybean ModernSurface sterilized seeds Bacillaceae Bacillus sp. SYM00697 41 745 RiceModern Seed surface wash Bacillaceae Bacillus sp. SYM00704 41 746 RiceModern Seed surface wash Bacillaceae Bacillus sp. SYM00017c 45 533 RiceModern Surface sterilized seeds Sphingomonadaceae Sphingomonas sp.SYM00062b 45 558 Teosinte Wild relative Surface sterilized seedsSphingomonadaceae Sphingomonas sp. SYM00065 45 561 Teosinte Wildrelative Surface sterilized seeds Sphingomonadaceae Sphingomonas sp.SYM00168 45 589 Rice Modern Surface sterilized seeds SphingomonadaceaeSphingomonas sp. SYM00169 45 590 Rice Modern Surface sterilized seedsSphingomonadaceae Sphingomonas sp. SYM00942 45 817 Rice Modern Surfacesterilized seeds Sphingomonadaceae Sphingomonas sp. SYM00994 45 858Oryza officinalis Wild relative Surface sterilized seedsSphingomonadaceae Sphingomonas sp. SYM01016 45 874 Rice Modern Surfacesterilized seeds Sphingomonadaceae Sphingomonas sp. SYM01174 45 944 RiceAncient Landrace Roots & Seeds Sphingomonadaceae Sphingomonas sp.SYM01176 45 946 Rice Ancient Landrace Roots & Seeds SphingomonadaceaeSphingomonas sp. SYM01187 45 956 Rice Modern Roots & SeedsSphingomonadaceae Sphingomonas sp. SYM01191 45 960 Rice Modern Roots &Seeds Sphingomonadaceae Sphingomonas sp. SYM01214 45 970 Rice ModernRoots & Seeds Sphingomonadaceae Sphingomonas sp. SYM01216 45 972 RiceModern Roots & Seeds Sphingomonadaceae Sphingomonas sp. SYM00231 46 620Soybean Modern Leaves Sphingomonadaceae Sphingobium sp. SYM00975 51 843Rice Ancient Landrace Surface sterilized seeds OxalobacteraceaeHerbaspirillum sp. SYM00506c 53 643 Maize Ancient Landrace Seed surfacewash Paenibacillaceae Paenibacillus sp. SYM00506D 53 644 Maize AncientLandrace Seed surface wash Paenibacillaceae Paenibacillus sp. SYM0054553 664 Maize Ancient Landrace Seed surface wash PaenibacillaceaePaenibacillus sp. SYM00549 53 668 Maize Ancient Landrace Seed surfacewash Paenibacillaceae Paenibacillus sp. SYM00554 53 672 Maize AncientLandrace Seed surface wash Paenibacillaceae Paenibacillus sp. SYM0055553 673 Maize Ancient Landrace Seed surface wash PaenibacillaceaePaenibacillus sp. SYM00012 55 1447 Teosinte Wild relative Surfacesterilized seeds Microbacteriaceae Microbacterium binotii SYM00046 56545 Maize Ancient Landrace Surface sterilized seeds EnterobacteriaceaeEnterobacter sp. SYM00050 56 548 Maize Ancient Landrace Surfacesterilized seeds Enterobacteriaceae Enterobacter sp. SYM00628 56 726Maize Modern Seed surface wash Enterobacteriaceae Enterobacter sp.SYM01049 56 898 Teosinte Wild relative Surface sterilized seedsEnterobacteriaceae SYM00106 59 583 Maize Ancient Landrace Surfacesterilized seeds Micrococcaceae Micrococcus sp. SYM00107 59 584 MaizeAncient Landrace Surface sterilized seeds Micrococcaceae Micrococcus sp.SYM00108 59 585 Maize Ancient Landrace Surface sterilized seedsMicrococcaceae Micrococcus sp. SYM00254 59 631 Maize Modern Surfacesterilized seeds Micrococcaceae Micrococcus sp. SYM00090 62 575 MaizeAncient Landrace Surface sterilized seeds FlavobacteriaceaeChryseobacterium sp. SYM00002 66 521 Teosinte Wild relative Surfacesterilized seeds Rhizobiaceae Agrobacterium sp. SYM00017a 66 531 RiceModern Surface sterilized seeds Rhizobiaceae Agrobacterium sp. SYM0032666 635 Maize Modern Roots Rhizobiaceae Agrobacterium tumefaciensSYM00714 66 750 Rice Modern Seed surface wash Rhizobiaceae Agrobacteriumsp. SYM00983 66 849 Rice Modern Surface sterilized seeds RhizobiaceaeAgrobacterium sp. SYM01004 66 865 Rice Modern Surface sterilized seedsRhizobiaceae Agrobacterium sp. SYM00060 67 556 Maize Ancient LandraceSurface sterilized seeds Staphylococcaceae Staphylococcus sp. SYM0011367 587 Maize Modern Surface sterilized seeds StaphylococcaceaeStaphylococcus sp. SYM01257 67 992 Rice Ancient Landrace Roots & SeedsStaphylococcaceae Staphylococcus sp. SYM01259 67 993 Rice AncientLandrace Roots & Seeds Staphylococcaceae Staphylococcus sp. SYM00071 76565 Teosinte Wild relative Surface sterilized seeds Bacillaceae Bacillussp. SYM00204 76 614 Maize Maize Roots Bacillaceae Bacillus sp. SYM0056376 678 Maize Ancient Landrace Seed surface wash Bacillaceae Bacillus sp.SYM00617 76 719 Teosinte Wild relative Seed surface wash BacillaceaeBacillus sp. SYM00016c 82 530 Rice Modern Surface sterilized seedsXanthomonadaceae Luteibacter sp. SYM00960 82 831 Rice Ancient LandraceSurface sterilized seeds Xanthomonadaceae Luteibacter sp. SYM00965 82835 Rice Ancient Landrace Surface sterilized seeds XanthomonadaceaeLuteibacter sp. SYM01167 82 941 Rice Ancient Landrace Roots & SeedsXanthomonadaceae Luteibacter sp. SYM00940 83 815 Rice Modern Surfacesterilized seeds Enterobacteriaceae SYM00941 83 816 Rice Modern Surfacesterilized seeds Enterobacteriaceae SYM00963 83 833 Rice AncientLandrace Surface sterilized seeds Enterobacteriaceae SYM00972 83 841Rice Ancient Landrace Surface sterilized seeds EnterobacteriaceaeSYM00987 83 853 Rice Modern Surface sterilized seeds EnterobacteriaceaeSYM00713 84 749 Rice Modern Seed surface wash Enterobacteriaceae Erwiniasp. SYM00945 84 820 Rice Modern Surface sterilized seedsEnterobacteriaceae SYM01103 84 914 Rice Modern Surface sterilized seedsEnterobacteriaceae SYM01138 84 926 Oryza latifolia Wild relative Roots &Seeds Enterobacteriaceae SYM01139 84 927 Oryza latifolia Wild relativeRoots & Seeds Enterobacteriaceae SYM01180 84 950 Rice Modern Roots &Seeds Enterobacteriaceae SYM01189 84 958 Rice Modern Roots & SeedsEnterobacteriaceae SYM01193 84 961 Rice Modern Roots & SeedsEnterobacteriaceae SYM01226 84 976 Rice Modern Roots & SeedsEnterobacteriaceae SYM01229 84 978 Rice Modern Roots & SeedsEnterobacteriaceae Pantoea sp. SYM01230 84 979 Rice Modern Roots & SeedsEnterobacteriaceae SYM00992 126 856 Oryza officinalis Wild relativeSurface sterilized seeds Sphingomonadaceae Sphingomonas sp. SYM00063 134559 Teosinte Wild relative Surface sterilized seeds MicrobacteriaceaeMicrobacterium sp. SYM00226 134 618 Soybean Modern LeavesMicrobacteriaceae Microbacterium sp. SYM00246 134 627 Barley ModernSurface sterilized seeds Microbacteriaceae Microbacterium sp. SYM00524134 653 Oryza nivara Wild relative Seed surface wash MicrobacteriaceaeMicrobacterium sp. SYM00694a 134 743 Rice Modern Seed surface washMicrobacteriaceae Microbacterium sp. SYM01234 134 983 Oryza officinalisWild relative Roots & Seeds Microbacteriaceae Microbacterium sp.SYM00199 135 1448 Maize Maize Roots Bacillaceae Bacillus sp. SYM00172146 592 Rice Modern Surface sterilized seeds Enterobacteriaceae Pantoeasp. SYM00527 146 655 Oryza nivara Wild relative Seed surface washEnterobacteriaceae Erwinia sp. SYM00644 146 728 Rice Modern Seed surfacewash Enterobacteriaceae Erwinia sp. SYM00648 146 732 Rice Modern Seedsurface wash Enterobacteriaceae SYM00966 146 836 Rice Ancient LandraceSurface sterilized seeds Enterobacteriaceae SYM00978 146 846 RiceAncient Landrace Surface sterilized seeds Enterobacteriaceae SYM00981146 848 Rice Modern Surface sterilized seeds Enterobacteriaceae SYM01011146 870 Rice Ancient Landrace Surface sterilized seedsEnterobacteriaceae Erwinia sp. SYM01159 146 938 Avena sterilis Wildrelative Roots & Seeds Enterobacteriaceae SYM01175 146 945 Rice AncientLandrace Roots & Seeds Enterobacteriaceae SYM01232 146 981 Rice ModernRoots & Seeds Enterobacteriaceae SYM01244 146 987 Rice Ancient LandraceRoots & Seeds Enterobacteriaceae SYM00538A 172 658 Maize AncientLandrace Seed surface wash Sphingomonadaceae Sphingomonas sp. SYM00508196 646 Maize Ancient Landrace Seed surface wash EnterobacteriaceaeLegend: For “Source of seed-origin microbe” “Surface sterilized seeds” =seed-origin microbes isolated from seeds that were surface sterilized asdescribed in the Examples; “Seed surface wash” = microbes derived fromthe surface of seeds as described in the Examples; “Roots” = seed-originmicrobes isolated from roots of seeds that were germinated in sterileculture; “Roots & Seeds” = seed-origin microbes isolated from roots andresidual seed material that was generated by germinating seeds understerile conditions; “Leaves” = seed-origin microbes isolated from shootsand leaves that emerged from seeds that were germinated under sterileconditions.

As used herein, seed-origin endophytes can be obtained from seeds ofmany distinct plants. In one embodiment, the endophyte can be obtainedfrom the seed of the same or different crop, and can be from the same ordifferent cultivar or variety as the seed onto which it is to be coated.For example, seed endophytes from a particular corn variety can beisolated and coated onto the surface of a corn seed of the same variety.In one particular embodiment, the seed of the first plant that is to becoated with the endophyte can comprise a detectable amount of the sameendophyte in the interior of the seed. In another embodiment, the seedof the first plant that is to be coated with the endophyte can comprisea detectable amount of the same endophyte in the exterior of the seed.For example, an uncoated reference seed may contain a detectable amountof the same endophyte within its seed. In yet another embodiment, theendophyte to be coated onto the seed of the plant is a microbe or of amicrobial taxa that is detectably present in the interior and exteriorof the seed from which the endophyte is derived.

In another embodiment, the endophyte can be obtained from a relatedspecies (e.g., an endophyte isolated from Triticum monococcum (einkornwheat) can be coated onto the surface of a T. aestivum (common wheat)seed; or, an endophyte from Hordeum vulgare (barley) can be isolated andcoated onto the seed of a member of the Triticeae family, for example,seeds of the rye plant, Secale cereale). In still another embodiment,the endophyte can be isolated from the seed of a plant that is distantlyrelated to the seed onto which the endophyte is to be coated. Forexample, a tomato-derived endophyte is isolated and coated onto a riceseed.

In some embodiments, the present invention contemplates the use ofendophytes that can confer a beneficial agronomic trait upon the seed orresulting plant onto which it is coated. In another embodiment, the seedendophytes useful for the present invention can also be isolated fromseeds of plants adapted to a particular environment, including, but notlimited to, an environment with water deficiency, salinity, acute and/orchronic heat stress, acute and/or chronic cold stress, nutrient deprivedsoils including, but not limited to, micronutrient deprived soils,macronutrient (e.g., potassium, phosphate, nitrogen) deprived soils,pathogen stress, including fungal, nematode, insect, viral, bacterialpathogen stress. In one example, the endophyte is isolated from the seedof a plant that grows in a water deficient environment.

The synthetic combination of the present invention contemplates thepresence of an endophyte on the surface of the seed of the first plant.In one embodiment, the seed of the first plant is coated with at least10 CFU of the endophyte per seed, for example, at least 20 CFU, at least50 CFU, at least 100 CFU, at least 200 CFU, at least 300 CFU, at least500 CFU, at least 1,000 CFU, at least 3,000 CFU, at least 10,000 CFU, atleast 30,000 or more per seed. In another embodiment, the seed is coatedwith at least 10, for example, at least 20, at least 50, at least 100,at least 200, at least 300, at least 500, at least 1,000, at least3,000, at least 10,000, at least 30,000, at least 100,000, at least300,000, at least 1,000,000 or more of the endophyte as detected by thenumber of copies of a particular endophyte gene detected, for example,by quantitative PCR.

In some cases, the seed-origin endophyte is of monoclonal origin,providing high genetic uniformity of the endophyte population in anagricultural formulation or within a synthetic seed or plant combinationwith the endophyte.

In some cases, the bacterial endophytes described herein are capable ofmoving from one tissue type to another. For example, the presentinvention's detection and isolation of seed-origin endophytes within themature tissues of cereal plants after coating on the exterior of a seeddemonstrates their ability to move from seed exterior into thevegetative tissues of a maturing plant. Therefore, in one embodiment,the population of bacterial endophytes is capable of moving from theseed exterior into the vegetative tissues of a grass plant. In oneembodiment, the seed endophyte which is coated onto the seed of a plantis capable, upon germination of the seed into a vegetative state, oflocalizing to a different tissue of the plant. For example, theendophyte can be capable of localizing to any one of the tissues in theplant, including: the root, adventitious root, seminal root, root hair,shoot, leaf, flower, bud, tassel, meristem, pollen, pistil, ovaries,stamen, fruit, stolon, rhizome, nodule, tuber, trichome, guard cells,hydathode, petal, sepal, glume, rachis, vascular cambium, phloem, andxylem. In one embodiment, the endophyte is capable of localizing to theroot and/or the root hair of the plant. In another embodiment, theendophyte is capable of localizing to the photosynthetic tissues, forexample, leaves and shoots of the plant. In other cases, the endophyteis localized to the vascular tissues of the plant, for example, in thexylem and phloem. In still another embodiment, the endophyte is capableof localizing to the reproductive tissues (flower, pollen, pistil,ovaries, stamen, fruit) of the plant. In another embodiment, theendophyte is capable of localizing to the root, shoots, leaves andreproductive tissues of the plant. In still another embodiment, theendophyte colonizes a fruit or seed tissue of the plant. In stillanother embodiment, the endophyte is able to colonize the plant suchthat it is present in the surface of the plant (i.e., its presence isdetectably present on the plant exterior, or the episphere of theplant). In still other embodiments, the endophyte is capable oflocalizing to substantially all, or all, tissues of the plant. Incertain embodiments, the endophyte is not localized to the root of aplant. In other cases, the endophyte is not localized to thephotosynthetic tissues of the plant.

In some cases, the bacterial endophytes are capable of replicatingwithin the host grass plant and colonizing the grass plant.

In addition, the bacterial endophytes described herein provide severalkey significant advantages over other plant-associated microbes.

Different environments can contain significantly different populationsof endophytes and thus may provide reservoirs for desired seed-originendophytes. Once a choice environment is selected, seeds of choiceplants to be sampled can be identified by their healthy and/or robustgrowth, and can then be sampled at least 5 at a time by excavating theentire plants plus small root ball including roots and associated soiland any seeds or fruit present on the plant. The excavated material canbe placed in a cool (4° C. environment) for storage, and then extractionof endophytes and DNA can be performed using methods described herein.Identification of choice environments or ecosystems for bioprospectingof plant associated endophytes from either wild plants or crop plantsgrowing in the choice environments or ecosystems follows protocolsdescribed herein.

In one embodiment, the endophyte-associated plant is harvested from asoil type different than the normal soil type that the crop plant isgrown on, for example from a gelisol (soils with permafrost within 2 mof the surface), for example from a histosol (organic soil), for examplefrom a spodosol (acid forest soils with a subsurface accumulation ofmetal-humus complexes), for example from an andisol (soils formed involcanic ash), for example from a oxisol (intensely weathered soils oftropical and subtropical environments), for example from a vertisol(clayey soils with high shrink/swell capacity), for example from anaridisol (CaCO₃-containing soils of arid environments with subsurfacehorizon development), for example from a ultisol (strongly leached soilswith a subsurface zone of clay accumulation and <35% base saturation),for example from a mollisol (grassland soils with high base status), forexample from an alfisol (moderately leached soils with a subsurface zoneof clay accumulation and >35% base saturation), for example from ainceptisol (soils with weakly developed subsurface horizons), or forexample from a entisol (soils with little or no morphologicaldevelopment).

In another embodiment, the endophyte-associated plant is harvested froman ecosystem where the agricultural plant is not normally found, forexample, a tundra ecosystem as opposed to a temperate agricultural farm,for example from tropical and subtropical moist broadleaf forests(tropical and subtropical, humid), for example from tropical andsubtropical dry broadleaf forests (tropical and subtropical, semihumid),for example from tropical and subtropical coniferous forests (tropicaland subtropical, semihumid), for example from temperate broadleaf andmixed forests (temperate, humid), for example from temperate coniferousforests (temperate, humid to semihumid), from for example from borealforests/taiga (subarctic, humid), for example from tropical andsubtropical grasslands, savannas, and shrublands (tropical andsubtropical, semiarid), for example from temperate grasslands, savannas,and shrublands (temperate, semiarid), for example from floodedgrasslands and savannas (temperate to tropical, fresh or brackish waterinundated), for example from montane grasslands and shrublands (alpineor montane climate), for example from Mediterranean forests, woodlands,and scrub or sclerophyll forests (temperate warm, semihumid to semiaridwith winter rainfall), for example from mangrove forests, and forexample from deserts and xeric shrublands (temperate to tropical, arid).

In another embodiment, the endophyte-associated plant is harvested froma soil with an average pH range that is different from the optimal soilpH range of the crop plant, for example the plant may be harvested froman ultra acidic soil (<3.5), from an extreme acid soil (3.5-4.4), from avery strong acid soil (4.5-5.0), from a strong acid soil (5.1-5.5), froma moderate acid soil (5.6-6.0), from an slight acid soil (6.1-6.5), froman neutral soil (6.6-7.3), from an slightly alkaline soil (7.4-7.8),from an moderately alkaline soil (7.9-8.4), from a strongly alkalinesoil (8.5-9.0), or from an very strongly alkaline soil (>9.0).

In one embodiment, the endophyte-associated plant is harvested from anenvironment with average air temperatures lower than the normal growingtemperature of the crop plant, for example 2-5° C. colder than average,for example, at least 5-10° C. colder, at least 10-15° C. colder, atleast at least 15-20° C. colder, at least 20-25° C. colder, at least25-30° C. colder, at least 30-35° C. colder, at least 35-40° C. colder,at least 40-45° C. colder, at least 45-50° C. colder, at least 50-55° C.colder or more, when compared with crop plants grown under normalconditions during an average growing season.

In one embodiment, the endophyte-associated plant is harvested from anenvironment with average air temperatures higher than the normal growingtemperature of the crop plant, for example 2-5° C. hotter than average,for example, at least 5-10° C. hotter, at least 10-15° C. hotter, atleast at least 15-20° C. hotter, at least 20-25° C. hotter, at least25-30° C. hotter, at least 30-35° C. hotter, at least 35-40° C. hotter,at least 40-45° C. hotter, at least 45-50° C. hotter, at least 50-55° C.hotter or more, when compared with crop plants grown under normalconditions during an average growing season.

In another embodiment, the endophyte-associated plant is harvested froman environment with average rainfall lower than the optimal averagerainfall received by the crop plant, for example 2-5% less rainfall thanaverage, for example, at least 5-10% less rainfall, at least 10-15% lessrainfall, at least 15-20% less rainfall, at least 20-25% less rainfall,at least 25-30% less rainfall, at least 30-35% less rainfall, at least35-40% less rainfall, at least 40-45% less rainfall, at least 45-50%less rainfall, at least 50-55% less rainfall, at least 55-60% lessrainfall, at least 60-65% less rainfall, at least 65-70% less rainfall,at least 70-75% less rainfall, at least 80-85% less rainfall, at least85-90% less rainfall, at least 90-95% less rainfall, or less, whencompared with crop plants grown under normal conditions during anaverage growing season.

In one embodiment, the endophyte-associated plant is harvested from anenvironment with average rainfall higher than the optimal averagerainfall of the crop plant, for example 2-5% more rainfall than average,for example, at least 5-10% more rainfall, at least 10-15% morerainfall, at least 15-20% more rainfall, at least 20-25% more rainfall,at least 25-30% more rainfall, at least 30-35% more rainfall, at least35-40% more rainfall, at least 40-45% more rainfall, at least 45-50%more rainfall, at least 50-55% more rainfall, at least 55-60% morerainfall, at least 60-65% more rainfall, at least 65-70% more rainfall,at least 70-75% more rainfall, at least 80-85% more rainfall, at least85-90% more rainfall, at least 90-95% more rainfall, at least 95-100%more rainfall, or even greater than 100% more rainfall, or even greaterthan 200% more rainfall, or even greater than 300% more rainfall, oreven greater than 400% more rainfall, or even greater than 500% morerainfall, when compared with crop plants grown under normal conditionsduring an average growing season.

In another embodiment, the endophyte-associated plant is harvested froma soil type with different soil moisture classification than the normalsoil type that the crop plant is grown on, for example from an aquicsoil (soil is saturated with water and virtually free of gaseous oxygenfor sufficient periods of time, such that there is evidence of pooraeration), for example from an udic soil (soil moisture is sufficientlyhigh year-round in most years to meet plant requirement), for examplefrom an ustic soil (soil moisture is intermediate between udic andaridic regimes; generally, plant-available moisture during the growingseason, but severe periods of drought may occur), for example from anaridic soil (soil is dry for at least half of the growing season andmoist for less than 90 consecutive days), for example from a xeric soil(soil moisture regime is found in Mediterranean-type climates, withcool, moist winters and warm, dry summers).

In one embodiment, the endophyte-associated plant is harvested from anenvironment with average rainfall lower than the optimal averagerainfall of the crop plant, for example 2-95% less rainfall thanaverage, for example, at least 5-90% less rainfall, at least 10-85% lessrainfall, at least 15-80% less rainfall, at least 20-75% less rainfall,at least 25-70% less rainfall, at least 30-65% less rainfall, at least35-60% less rainfall, at least 40-55% less rainfall, at least 45-50%less rainfall, when compared with crop plants grown under normalconditions during an average growing season.

In one embodiment, the endophyte-associated plant is harvested from anenvironment with average rainfall higher than the optimal averagerainfall of the crop plant, for example 2-5% more rainfall than average,for example, at least 5-10% more rainfall, at least 10-15% morerainfall, at least 15-20% more rainfall, at least 20-25% more rainfall,at least 25-30% more rainfall, at least 30-35% more rainfall, at least35-40% more rainfall, at least 40-45% more rainfall, at least 45-50%more rainfall, at least 50-55% more rainfall, at least 55-60% morerainfall, at least 60-65% more rainfall, at least 65-70% more rainfall,at least 70-75% more rainfall, at least 80-85% more rainfall, at least85-90% more rainfall, at least 90-95% more rainfall, at least 95-100%more rainfall, or even greater than 100% more rainfall, or even greaterthan 200% more rainfall, or even greater than 300% more rainfall, oreven greater than 400% more rainfall, or even greater than 500% morerainfall, when compared with crop plants grown under normal conditionsduring an average growing season.

In another embodiment, the endophyte-associated plant is harvested froman agricultural environment with a crop yield lower than the averagecrop yield expected from the crop plant grown under average cultivationpractices on normal agricultural land, for example 2-5% lower yield thanaverage, for example, at least 5-10% lower yield, at least 10-15% loweryield, at least 15-20% lower yield, at least 20-25% lower yield, atleast 25-30% lower yield, at least 30-35% lower yield, at least 35-40%lower yield, at least 40-45% lower yield, at least 45-50% lower yield,at least 50-55% lower yield, at least 55-60% lower yield, at least60-65% lower yield, at least 65-70% lower yield, at least 70-75% loweryield, at least 80-85% lower yield, at least 85-90% lower yield, atleast 90-95% lower yield, or less, when compared with crop plants grownunder normal conditions during an average growing season.

In a related embodiment, the endophyte-associated plant is harvestedfrom an agricultural environment with a crop yield lower than theaverage crop yield expected from the crop plant grown under averagecultivation practices on normal agricultural land, for example 2-95%lower yield than average, for example, at least 5-90% lower yield, atleast 10-85% lower yield, at least 15-80% lower yield, at least 20-75%lower yield, at least 25-70% lower yield, at least 30-65% lower yield,at least 35-60% lower yield, at least 40-55% lower yield, at least45-50% lower yield, when compared with crop plants grown under normalconditions during an average growing season.

In one embodiment, the endophyte-associated plant is harvested from anenvironment with average crop yield higher than the optimal average cropyield of the crop plant, for example 2-5% more yield than average, forexample, at least 5-10% more yield, at least 10-15% more yield, at least15-20% more yield, at least 20-25% more yield, at least 25-30% moreyield, at least 30-35% more yield, at least 35-40% more yield, at least40-45% more yield, at least 45-50% more yield, at least 50-55% moreyield, at least 55-60% more yield, at least 60-65% more yield, at least65-70% more yield, at least 70-75% more yield, at least 80-85% moreyield, at least 85-90% more yield, at least 90-95% more yield, at least95-100% more yield, or even greater than 100% more yield, or evengreater than 200% more yield, or even greater than 300% more yield, oreven greater than 400% more yield, or even greater than 500% more yield,when compared with crop plants grown under normal conditions during anaverage growing season.

In a related embodiment, the endophyte-associated plant is harvestedfrom an environment with average crop yield higher than the optimalaverage crop yield of the crop plant, 2-500% more yield than average,2-400% more yield than average, 2-300% more yield than average, 2-200%more yield than average, 2-95% more yield than average, for example, atleast 5-90% more yield, at least 10-85% more yield, at least 15-80% moreyield, at least 20-75% more yield, at least 25-70% more yield, at least30-65% more yield, at least 35-60% more yield, at least 40-55% moreyield, at least 45-50% more yield, when compared with crop plants grownunder normal conditions during an average growing season.

In another embodiment, the endophyte-associated plant is harvested froman environment where soil contains lower total nitrogen than the optimumlevels recommended in order to achieve average crop yields for a plantgrown under average cultivation practices on normal agricultural land,for example 2-5% less nitrogen than average, for example, at least 5-10%less nitrogen, at least 10-15% less nitrogen, at least 15-20% lessnitrogen, at least 20-25% less nitrogen, at least 25-30% less nitrogen,at least 30-35% less nitrogen, at least 35-40% less nitrogen, at least40-45% less nitrogen, at least 45-50% less nitrogen, at least 50-55%less nitrogen, at least 55-60% less nitrogen, at least 60-65% lessnitrogen, at least 65-70% less nitrogen, at least 70-75% less nitrogen,at least 80-85% less nitrogen, at least 85-90% less nitrogen, at least90-95% less nitrogen, or less, when compared with crop plants grownunder normal conditions during an average growing season.

In another embodiment, the endophyte-associated plant is harvested froman environment where soil contains higher total nitrogen than theoptimum levels recommended in order to achieve average crop yields for aplant grown under average cultivation practices on normal agriculturalland, for example 2-5% more nitrogen than average, for example, at least5-10% more nitrogen, at least 10-15% more nitrogen, at least 15-20% morenitrogen, at least 20-25% more nitrogen, at least 25-30% more nitrogen,at least 30-35% more nitrogen, at least 35-40% more nitrogen, at least40-45% more nitrogen, at least 45-50% more nitrogen, at least 50-55%more nitrogen, at least 55-60% more nitrogen, at least 60-65% morenitrogen, at least 65-70% more nitrogen, at least 70-75% more nitrogen,at least 80-85% more nitrogen, at least 85-90% more nitrogen, at least90-95% more nitrogen, at least 95-100% more nitrogen, or even greaterthan 100% more nitrogen, or even greater than 200% more nitrogen, oreven greater than 300% more nitrogen, or even greater than 400% morenitrogen, or even greater than 500% more nitrogen, when compared withcrop plants grown under normal conditions during an average growingseason.

In another embodiment, the endophyte-associated plant is harvested froman environment where soil contains lower total phosphorus than theoptimum levels recommended in order to achieve average crop yields for aplant grown under average cultivation practices on normal agriculturalland, for example 2-5% less phosphorus than average, for example, atleast 5-10% less phosphorus, at least 10-15% less phosphorus, at least15-20% less phosphorus, at least 20-25% less phosphorus, at least 25-30%less phosphorus, at least 30-35% less phosphorus, at least 35-40% lessphosphorus, at least 40-45% less phosphorus, at least 45-50% lessphosphorus, at least 50-55% less phosphorus, at least 55-60% lessphosphorus, at least 60-65% less phosphorus, at least 65-70% lessphosphorus, at least 70-75% less phosphorus, at least 80-85% lessphosphorus, at least 85-90% less phosphorus, at least 90-95% lessphosphorus, or less, when compared with crop plants grown under normalconditions during an average growing season.

In another embodiment, the endophyte-associated plant is harvested froman environment where soil contains higher total phosphorus than theoptimum levels recommended in order to achieve average crop yields for aplant grown under average cultivation practices on normal agriculturalland, for example 2-5% more phosphorus than average, for example, atleast 5-10% more phosphorus, at least 10-15% more phosphorus, at least15-20% more phosphorus, at least 20-25% more phosphorus, at least 25-30%more phosphorus, at least 30-35% more phosphorus, at least 35-40% morephosphorus, at least 40-45% more phosphorus, at least 45-50% morephosphorus, at least 50-55% more phosphorus, at least 55-60% morephosphorus, at least 60-65% more phosphorus, at least 65-70% morephosphorus, at least 70-75% more phosphorus, at least 80-85% morephosphorus, at least 85-90% more phosphorus, at least 90-95% morephosphorus, at least 95-100% more phosphorus, or even greater than 100%more phosphorus, or even greater than 200% more phosphorus, or evengreater than 300% more phosphorus, or even greater than 400% morephosphorus, or even greater than 500% more phosphorus, when comparedwith crop plants grown under normal conditions during an average growingseason.

In another embodiment, the endophyte-associated plant is harvested froman environment where soil contains lower total potassium than theoptimum levels recommended in order to achieve average crop yields for aplant grown under average cultivation practices on normal agriculturalland, for example 2-5% less potassium than average, for example, atleast 5-10% less potassium, at least 10-15% less potassium, at least15-20% less potassium, at least 20-25% less potassium, at least 25-30%less potassium, at least 30-35% less potassium, at least 35-40% lesspotassium, at least 40-45% less potassium, at least 45-50% lesspotassium, at least 50-55% less potassium, at least 55-60% lesspotassium, at least 60-65% less potassium, at least 65-70% lesspotassium, at least 70-75% less potassium, at least 80-85% lesspotassium, at least 85-90% less potassium, at least 90-95% lesspotassium, or less, when compared with crop plants grown under normalconditions during an average growing season.

In another embodiment, the endophyte-associated plant is harvested froman environment where soil contains higher total potassium than theoptimum levels recommended in order to achieve average crop yields for aplant grown under average cultivation practices on normal agriculturalland, for example 2-5% more potassium than average, for example, atleast 5-10% more potassium, at least 10-15% more potassium, at least15-20% more potassium, at least 20-25% more potassium, at least 25-30%more potassium, at least 30-35% more potassium, at least 35-40% morepotassium, at least 40-45% more potassium, at least 45-50% morepotassium, at least 50-55% more potassium, at least 55-60% morepotassium, at least 60-65% more potassium, at least 65-70% morepotassium, at least 70-75% more potassium, at least 80-85% morepotassium, at least 85-90% more potassium, at least 90-95% morepotassium, at least 95-100% more potassium, or even greater than 100%more potassium, or even greater than 200% more potassium, or evengreater than 300% more potassium, or even greater than 400% morepotassium, or even greater than 500% more potassium, when compared withcrop plants grown under normal conditions during an average growingseason.

In another embodiment, the endophyte-associated plant is harvested froman environment where soil contains lower total sulfur than the optimumlevels recommended in order to achieve average crop yields for a plantgrown under average cultivation practices on normal agricultural land,for example 2-5% less sulfur than average, for example, at least 5-10%less sulfur, at least 10-15% less sulfur, at least 15-20% less sulfur,at least 20-25% less sulfur, at least 25-30% less sulfur, at least30-35% less sulfur, at least 35-40% less sulfur, at least 40-45% lesssulfur, at least 45-50% less sulfur, at least 50-55% less sulfur, atleast 55-60% less sulfur, at least 60-65% less sulfur, at least 65-70%less sulfur, at least 70-75% less sulfur, at least 80-85% less sulfur,at least 85-90% less sulfur, at least 90-95% less sulfur, or less, whencompared with crop plants grown under normal conditions during anaverage growing season.

In another embodiment, the endophyte-associated plant is harvested froman environment where soil contains higher total sulfur than the optimumlevels recommended in order to achieve average crop yields for a plantgrown under average cultivation practices on normal agricultural land,for example 2-5% more sulfur than average, for example, at least 5-10%more sulfur, at least 10-15% more sulfur, at least 15-20% more sulfur,at least 20-25% more sulfur, at least 25-30% more sulfur, at least30-35% more sulfur, at least 35-40% more sulfur, at least 40-45% moresulfur, at least 45-50% more sulfur, at least 50-55% more sulfur, atleast 55-60% more sulfur, at least 60-65% more sulfur, at least 65-70%more sulfur, at least 70-75% more sulfur, at least 80-85% more sulfur,at least 85-90% more sulfur, at least 90-95% more sulfur, at least95-100% more sulfur, or even greater than 100% more sulfur, or evengreater than 200% more sulfur, or even greater than 300% more sulfur, oreven greater than 400% more sulfur, or even greater than 500% moresulfur, when compared with crop plants grown under normal conditionsduring an average growing season.

In another embodiment, the endophyte-associated plant is harvested froman environment where soil contains lower total calcium than the optimumlevels recommended in order to achieve average crop yields for a plantgrown under average cultivation practices on normal agricultural land,for example 2-5% less calcium than average, for example, at least 5-10%less calcium, at least 10-15% less calcium, at least 15-20% lesscalcium, at least 20-25% less calcium, at least 25-30% less calcium, atleast 30-35% less calcium, at least 35-40% less calcium, at least 40-45%less calcium, at least 45-50% less calcium, at least 50-55% lesscalcium, at least 55-60% less calcium, at least 60-65% less calcium, atleast 65-70% less calcium, at least 70-75% less calcium, at least 80-85%less calcium, at least 85-90% less calcium, at least 90-95% lesscalcium, or less, when compared with crop plants grown under normalconditions during an average growing season.

In another embodiment, the endophyte-associated plant is harvested froman environment where soil contains lower total magnesium than theoptimum levels recommended in order to achieve average crop yields for aplant grown under average cultivation practices on normal agriculturalland, for example 2-5% less magnesium than average, for example, atleast 5-10% less magnesium, at least 10-15% less magnesium, at least15-20% less magnesium, at least 20-25% less magnesium, at least 25-30%less magnesium, at least 30-35% less magnesium, at least 35-40% lessmagnesium, at least 40-45% less magnesium, at least 45-50% lessmagnesium, at least 50-55% less magnesium, at least 55-60% lessmagnesium, at least 60-65% less magnesium, at least 65-70% lessmagnesium, at least 70-75% less magnesium, at least 80-85% lessmagnesium, at least 85-90% less magnesium, at least 90-95% lessmagnesium, or less, when compared with crop plants grown under normalconditions during an average growing season.

In another embodiment, the endophyte-associated plant is harvested froman environment where soil contains higher total sodium chloride (salt)than the optimum levels recommended in order to achieve average cropyields for a plant grown under average cultivation practices on normalagricultural land, for example 2-5% more salt than average, for example,at least 5-10% more salt, at least 10-15% more salt, at least 15-20%more salt, at least 20-25% more salt, at least 25-30% more salt, atleast 30-35% more salt, at least 35-40% more salt, at least 40-45% moresalt, at least 45-50% more salt, at least 50-55% more salt, at least55-60% more salt, at least 60-65% more salt, at least 65-70% more salt,at least 70-75% more salt, at least 80-85% more salt, at least 85-90%more salt, at least 90-95% more salt, at least 95-100% more salt, oreven greater than 100% more salt, or even greater than 200% more salt,or even greater than 300% more salt, or even greater than 400% moresalt, or even greater than 500% more salt, when compared with cropplants grown under normal conditions during an average growing season.

Plants Useful for the Present Invention

In some embodiments, a bacterial endophyte of seed-origin can beintroduced into an agricultural grass plant, i.e., a plant of the familyGraminae (grasses). The grass plants into which the bacterial endophyteof seed-origin can be introduced may be any of the useful grassesbelonging to the genera Agropyron, Agrostis, Andropogon, Anthoxanthum,Arrhenatherum, Avena, Brachypodium, Bromus, Chloris, Cynodon, Dactylis,Elymus, Eragrostis, Festuca, Glyceria, Hierochloe, Hordeum, Lolium,Oryza, Panicum, Paspalum, Phalaris, Phleum, Poa, Setaria, Sorghum,Triticum, Zea and Zoysia.

In another embodiment, the target plant is selected from the wheats,including, Triticum monococcum, Triticum durum, Triticum turgidum,Triticum timopheevi (Timopheevs Wheat) and Triticum aestivum (BreadWheat).

In another embodiment, the target plant is a corn of the genus Zea. Zeais a genus of the family Graminae (Poaceae), commonly known as the grassfamily. The genus consists of some four species: Zea mays, cultivatedcorn and teosinte; Zea diploperennis Iltis et at., diploperennialteosinte; Zea luxurians (Durieu et Asch.) Bird; and Zea perennis(Hitchc.) Reeves et Mangelsd., perennial teosinte.

Accordingly, in one embodiment, the plant is selected from the group ofGraminae (grasses), including grasses of the genera Agropyron, Agrostis,Andropogon, Anthoxanthum, Arrhenatherum, Avena, Brachypodium, Bromus,Chloris, Cynodon, Dactylis, Elymus, Eragrostis, Festuca, Glyceria,Hierochloe, Hordeum, including Hordeum vulgare L., Hordeum distichon L.,and Hordeum irregulare, Lolium, Oryza, Panicum, Paspalum, Phalaris,Phleum, Poa, Setaria, Sorghum, Triticum, Zea, especially Zea mays,cultivated corn and teosinte, Zea diploperennis Iltis et at.,diploperennial teosinte, Zea luxurians (Durieu et Asch.) Bird; and Zeaperennis (Hitchc.) Reeves et Mangelsd., perennial teosinte, and Zoysia;wheats, including Triticum monococcum, Triticum turgidum, Triticumtimopheevi (Timopheevs Wheat) and Triticum aestivum (Bread Wheat); ryegrasses and bluegrasses, especially Kentucky bluegrass, Canadabluegrass, rough meadow grass, bulbous meadow grass, alpine meadowgrass, wavy meadow grass, wood meadow grass, Balforth meadow grass,swamp meadow grass, broad leaf meadow grass, narrow leaf meadow grass,smooth meadow grass, spreading meadow grass and flattened meadow grass.

Commercial cultivars of agricultural plants can be used in the methodsand compositions as described herein. Non-limiting examples ofcommercial cultivars are provided below.

Maize

Exemplary Zea cultivars provided herein include 39V07, 38H03AM1, P9675,P9675YXR, P9630AM1, P9990AM1, P9917, P9917AM1, P9910AM1, P9910AMRW,P9910AMX, P9910XR, P0062AMX, P0062XR, P0193AM, P0193HR, P0216HR,P0210HR, 36V51, 36V52, 36V53, 36V59, P0313AM1, P0313XR, P0463AM1,P0461AMX, P0461EXR, P0461XR, P0453AM, P0453HR, P0448, P0448AMRW,P0448AMX, P0448E, P0448EHR, P0448R, P0413AM1, P0413E, P0407AMXT,P0533AM1, P0533EXR, P0528AMX, P0528YXR, 35F40, P0652AMX, P0636AM1,P0636HR, P0621HR, P0621R, P0717HR, P0832AM1, P0832E, P0832EXR, P0832XR,34F29, P0993AM1, P0993HR, P0993XR, P0987AM1, P0987HR, P0916EHR, 34R6,7P1023AM-R, P1018EHR, P1018HR, 34F06, 34F07, P1184, P1162AM1,P1162AMRW-R, P1162AMX-R, P1162EXR, P1162XR, P1151 AM, P1151AM1, P1151R,P1142AMX, 33W80, 33W82, 33W84, 33W88AM1, P1281HR, P1253E, P1248AM,P1221AMX, P1221AMXT, P1215AM1, P1395, P1395AM1, P1395HR, P1395R,P1376XR, P1365AMX, P1360CHR, P1360HR, P1339AM1, P1324HR, 33Z74, 33T56,33T57, 33M16, P1498, P1498AM, P1498HR, P1498R, P1480HR, P1477WHR,P1431W, P1431WR, P1420HR, 33G61, 33F12, P1555CHR, 33D42, 33D46, 33D49,P1659W, P1659WHR, 32D78, P1745HR, 32B16, P1995W, and P2088HR fromPioneer Hi-Bred, which are grown in geographical entities includingIowa. Exemplary Zea cultivars provided herein include P0115AM1,P0392AMX, P0496AMX, P0432AM1, P0413AM1, P0413AMRW, P0413E, P0413R,P0533AM1, P0636AM1, P0636YXR, 35K01, 35K02, 35K08, 35K09AM1, 35K10AMRW,34M78, P0858AMX, P0832AMRW, P0832AMX, P0832E, P0832EXR, P0832R,P0993AM1, P0993HR, P0987AM1, P0987YXR, P0945YXR, P0916EHR, 34R65,P1023AM-R, P1023AMX-R, P1018AM, P1018AM1, P1018AMX, P1018E, P1018R,P1184, P1184AM, P1184AM1, P1184AMRW, P1184R, P1162AM1, P1162AMRW-R,P1162AMX-R, P1162EXR, P1151AM, P1151AM1, 34P91, P1292AMX, P1241AMX,P1221AMX, P1221AMXT, P1215AM1, P1395AM1, P1395AMRW, P1376XR, P1360CHR,P1360HR, P1352AMX, P1339AM1, P1319, P1319AM1, P1319HR, 33T55, 33T56,P1498, P1498AM, P1498CHR, P1498HR, P1498R, P1477W, P1477WHR, P1449XR,P1431W, P1431WR, 33F12, 33D42, P1690HR, P1659W, 32B09, 32B10, 32B16,P1995W, P1995WR, and P2088AM from Pioneer Hi-Bred, which are grown ingeographical entities including Illinois.

Exemplary Zea cultivars provided herein include P8917XR, P9690AM,P9690HR, P0125R, P0231HR, P0365YHR, P0302CHR, P0474AM1, P0461EXR,P0591AM1, P0541AM1, P0541HR, 35F37, 35F38, 35F48AM1, 35F50AM, P0636AM1,P0636HR, P0636YXR, P0621HR, 35K01, P0876AM, P0876CHR, P0876HR, P0987,P0987AM, P0987AM1, P0987HR, P0987R, P0987YXR, P0916EHR, P0902AM1,P1023AM-R, P1023AMX-R, P1018EHR, P1173AM, P1173CHR, P1173HR, P1173R,P1151AM, P1151AM1, P1151HR, P1151R, P1151YXR, P1105YHR, P1292ER,P1266YHR, P1395AM, P1395AM1, P1395R, P1376XR, P1360HR, P1324HR, P1498AM,P1498AM1, P1498HR, P1498R, P1477W, P1477WHR, P1449XR, P1431W, 33G60,33G61, 33F12, P1508CHR, 32T16, 33D42, 33D46, 33D47, 33D49, 33D53AM-R,32T82, 32T84, P1690AM, P1690CHR, P1690HR, P1659W, P1659WHR, P1625CHR,P1625HR, P1768AMX, 32N74AM1, 32B09, 32B10, 32B11, 32B16, P1995W,P1995WR, 31G67AM1, 31G71, P2088AM, P2088YHR, and P2088YXR from PioneerHi-Bred, which are grown in geographical entities including Nebraska.

Exemplary Zea cultivars provided herein include P9690HR, P0115AM1,P0216HR, P0448E, P0432AM1, P0413AM1, P0413E, P0636AM1, P0636HR,P0636YHR, P0621HR, 35K01, 35K02, 35K08, 35K09AM1, 35K10AMRW, 34M78,P0858AMX, P0832AMX, P0832E, P0832R, P0993AM1, P0993HR, P0987, P0987AM,P0987AM1, P0987HR, P0987YXR, P0945YXR, P0916EHR, P1023AM-R, P1023AMX-R,P1018AM, P1018AM1, P1018AMX, P1018E, P1018R, P1184, P1184AM, P1184AM1,P1184R, P1162AM1, P1162AMRW-R, P1162AMX-R, P1151AM, P1151AM1, P1105YHR,34P91, P1253E, P1221AMX, P1221AMXT, P1395, P1395AMRW, P1395HR, P1395R,P1376XR, P1360AM, P1360HR, P1352AMX, P1339AM1, P1319, P1319AM1, P1319HR,33T54, 33T55, 33T56, 33T57, 33N58, P1498, P1498AM, P1498CHR, P1498HR,P1498R, P1477W, P1477WHR, P1449XR, P1431W, P1431WR, 33G60, 33F12,P1659W, P1659WHR, P1646YHR, P1636AM, P1636YHR, P1602YHR, 32D78, 32D79,P1745HR, 32B09, 32B10, 32B16, P1995W, P1995WR, 31P41, and P2088AM fromPioneer Hi-Bred, which are grown in geographical entities includingIndiana.

Exemplary Zea cultivars provided herein include Gentry® SmartStax® RIBComplete®, including DKC48-12RIB Brand, DKC49-29RIB Brand, DKC53-56RIBBrand, DKC62-08RIB Brand, DKC63-33RIB Brand; DEKALB® Genuity®DroughtGard™ Hybrids, including DKC47-27RIB Brand, DKC50-57RIB Brand,DKC51-20RIB Brand, DKC63-55RIB Brand, DKC65-81RIB Brand; <89 RelativeMaturity, including DKC31-10RIB Brand, DKC32-92RIB Brand, DKC33-78RIBBrand, DKC38-03RIB Brand, DKC39-07RIB Brand; 90-99 Relative Maturity,including DKC43-10RIB Brand, DKC44-13RIB Brand, DKC46-20RIB Brand,DKC48-12RIB Brand, DKC49-29RIB Brand; 101-103 Relative Maturity,including DKC51-20RIB Brand, DKC52-30RIB Brand, DKC53-56RIB Brand,DKC53-58RIB Brand, DKC53-78RIB Brand; 104-108 Relative Maturity,including DKC54-38RIB Brand, DKC57-75RIB Brand, DKC57-92RIB Brand,DKC58-87RIB Brand, DKC58-89RIB Brand; 110-111 Relative Maturity,including DKC60-63RIB Brand, DKC60-67RIB Brand, DKC61-16RIB Brand,DKC61-88RIB Brand, DKC61-89RIB Brand; 112-113 Relative Maturity,including DKC62-08RIB Brand, DKC62-97RIB Brand, DKC63-07RIB Brand,DKC63-33RIB Brand, DKC63-55RIB Brand; 114-116 Relative Maturity,including DKC64-69RIB Brand, DKC64-87RIB Brand, DKC65-19RIB Brand,DKC65-79RIB Brand, DKC66-40RIB Brand; 117+ Relative Maturity, includingDKC67-57RIB Brand, DKC67-58RIB Brand, DKC67-88RIB Brand, DKC68-05 Brand,and DKC69-29 Brand from DEKALB®, which are grown in geographicalentities including the United States.

Wheat

Exemplary Triticum cultivars provided herein include Everest, TAM 111,Armour, TAM 112, Fuller, Duster, T158, Postrock, Endurance, Jagger,Winter Hawk, Art, Overley, Jagalene, Jackpot, Hatcher, Santa Fe, Danby,Billings, T81, TAM 110, AP503 CL2, Aspen, 2137, TAM 113, Hitch, TAM 101,CJ, Centerfield, SY Gold, and Above, which are grown in geographicalentities including Kansas.

Exemplary Triticum cultivars provided herein include Barlow, Glenn, SYScren, Faller, Prosper, Kelby, Brennan, RB07, Vantage, WB Mayville,Freyr, Jenna, Mott, Select, Steele-ND, Briggs, Howard, Reeder, Alsen,Rollag, Divide, Alkabo, Mountrail, Tioga, Lebsock, Grenora, Dilse, Ben,DG Max, Pierce, Monroe, DG Star, Jerry, Decade, Hawken, Wesley,Overland, CDC Falcon, SY Wolf, Harding, Darrell, WB Matlock, Millennium,and Boomer, which are grown in geographical entities including N.Dakota.

Exemplary Triticum cultivars provided herein include Yellowstone, Genou,CDC Falcon, Rampart, Ledger, Jerry, AP503 CL2, Hawken, Norris, Pryor,Jagalene, Carter, Morgan, Decade, WB Quake, Tiber, Willow Creek,Radiant, Neeley, Vanguard, Promontory, Overland, and Redwin, which aregrown in geographical entities including Montana.

Exemplary Triticum cultivars provided herein include Duster, Endurance,Jagger, Fuller, OK Bullet, Jackpot, Everest, Billings, TAM 112, TAM 111,Big Max, Overley, Doans, Armour, Santa Fe, Garrison, Deliver, TAM 110,CJ, 2157, Custer, 2137, Scout, Centerfield, Triumph varieties, Dumas,TAM 401, Gallagher, Cutter, T-158, Ike, WB Hitch, Greer, AP 503 CL2,Ruby Lee, Pioneer 2548, Pioneer 2571, and Coker 762, which are grown ingeographical entities including Oklahoma.

Exemplary Triticum cultivars provided herein include UI Stone, Diva,Petit, Jubilee, Louise, Alturas, Whit, Babe, Cataldo, Alpowa,BrundageCF, Brundage96, Bitterroot, Kaseberg, Amber, Bruneau, AP Legacy,Salute, Ladd, Junction, ORCF101, Mary, Masami, SY Ovation, Skiles, Rod,WB523, Legion, Eltan, WB528, Stephens, Otto, ORCF103, Rosalyn, Madsen,AP Badger, LCS Artdeco, ORCF102, Lambert, Goetze, WB456, WB1020M,AP700CL, Xerpha, Tubbs06, WB1066CL, Eddy, Finley, Juniper, Whetstone,Sprinter1, Paladin, DW, Buchanan, Farnum, Northwest 553, Peregrine,Rimrock, Declo, Esperia, Boundary, Bauermeister, Residence, Symphony,and Estica, which are grown in geographical entities includingWashington state.

Exemplary Triticum cultivars provided herein include Wesley, Overland,Expedition, Clearfield, Smoky Hill, Arapahoe, Lyman, Hawken, Millennium,Jagalene, CDC Falcon, Alliance, Nekota, Briggs, RB07, Brick, Faller,Howard, Select, Traverse, Steele ND, Forge, Barlow, Butte86/Butte,Granger, Brennan, which are grown in geographical entities includingSouth Dakota.

Barley

Exemplary barley cultivars provided herein include Azure, Beacon, Bere,Betzes, Bowman, Celebration, Centennial, Compana, Conlon, Diamant,Dickson, Drummond, Excel, Foster, Glenn, Golden Promise, Hazen, Highlandbarley, Kindred, Kindred L, Larker, Logan, Lux, Manchurian, Manscheuri,Mansury, Maris Otter, Morex, Nordal, Nordic, Optic, Park, PlumageArcher, Pearl, Pinnacle, Proctor, Pioneer, Rawson, Robust, Sioux, Stark,Tradition, Traill, Tregal, Trophy, Windich, and Yagan, which are grownthroughout the world.

Exemplary barley cultivars provided herein include Tradition, Lacey,Robust, Celebration, Conlon, Pinnacle, Haybet, Legacy, Stellar-D,Innovation, Hays, Quest, Bowman, and Logan, which are grown ingeographical entities including North Dakota.

Exemplary barley cultivars provided herein include AC METCALFE,HARRINGTON, CONRAD (B5057), LEGACY (B2978), MORAVIAN 69 (C69), MERIT(B4947), TRADITION (B2482), MORAVIAN 83 (C83), and CHARLES, which aregrown in geographical entities including Idaho.

Exemplary barley cultivars provided herein include Harrington, Haybet, B1202, Moravian, Baronesse, Hector, Bowman, Westford, B Merit, Gallatin,Horsford, Lewis, Stark, Piroline, Valier, B 2601, Legacy, Menuet,Robust, Chinook, and Clark, which are grown in geographical entitiesincluding Montana.

Exemplary barley cultivars provided herein include Champion, Bob,Baronesse, Radiant, Haybet, Belford, Camelot, BG, Camas, Gallatin,Copeland, AC Metcalfe, and Harrington, which are grown in geographicalentities including Washington state.

Exemplary barley cultivars provided herein include Moravian 69, C-115,C-128, Scarlett, Baronesse, Hays, and Steptoe, which are grown ingeographical entities including Colorado.

Transgenic Plants

The methods described herein can also be used with transgenic plantscontaining one or more exogenous transgenes, for example, to yieldadditional trait benefits conferred by the newly introduced endophyticmicrobes. Therefore, in one embodiment, a seed or seedling of atransgenic maize, wheat, rice, or barley plant is contacted with anendophytic microbe.

Methods of Using Seed-Origin Bacterial Endophytes

As described herein, purified bacterial populations that include one ormore seed-origin bacterial endophytes and compositions containing thesame (e.g., agricultural formulations) can be used to confer beneficialtraits to the host plant including, for example, one or more of thefollowing: increased root biomass, increased root length, increasedheight, increased shoot length, increased leaf number, increased wateruse efficiency, increased overall biomass, increase grain yield,increased photosynthesis rate, increased tolerance to drought, increasedheat tolerance, increased salt tolerance, increased resistance tonematode stress, increased resistance to a fungal pathogen, increasedresistance to a bacterial pathogen, increased resistance to a viralpathogen, a detectable modulation in the level of a metabolite, and adetectable modulation in the proteome relative to a reference plant. Forexample, in some embodiments, a purified bacterial population thatincludes a seed-origin bacterial endophyte can improve two or more suchbeneficial traits, e.g., water use efficiency and increased tolerance todrought. Such traits can be heritable by progeny of the agriculturalplant to which the seed-origin bacterial endophyte was applied or byprogeny of the agricultural plant that was grown from the seedassociated with the seed-origin bacterial endophyte,

In some cases, the seed-origin bacterial endophyte may produce one ormore compounds and/or have one or more activities that are beneficial tothe plant, e.g., one or more of the following: production of ametabolite, production of a phytohormone such as auxin, production ofacetoin, production of an antimicrobial compound, production of asiderophore, production of a cellulase, production of a pectinase,production of a chitinase, production of a xylanase, nitrogen fixation,or mineral phosphate solubilization, For example, a seed-originbacterial endophyte can producea phytohormone selected from the groupconsisting of an auxin, a cytokinin, a gibberellin, ethylene, abrassinosteroid, and abscisic acid. In one particular embodiment, theseed-origin bacterial endophyte produces auxin (e.g., indole-3-aceticacid (IAA)). Production of auxin can be assayed as described herein.Many of the microbes described herein are capable of producing the planthormone auxin indole-3-acetic acid (IAA) when grown in culture. Auxinplays a key role in altering the physiology of the plant, including theextent of root growth. Therefore, in another embodiment, the bacterialendophytic population is disposed on the surface or within a tissue ofthe seed or seedling in an amount effective to detectably induceproduction of auxin in the agricultural plant. For example, the increasein auxin production can be at least 10%, for example, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 75%, atleast 100%, or more, when compared with a reference agricultural plant.In one embodiment, the increased auxin production can be detected in atissue type selected from the group consisting of the root, shoot,leaves, and flowers.

In some embodiments, the seed-origin bacterial endophyte can produce acompound with antimicrobial properties. For example, the compound canhave antibacterial properties, as determined by the growth assaysprovided herein. In one embodiment, the compound with antibacterialproperties shows bacteriostatic or bactericidal activity against E. coliand/or Bacillus sp. In another embodiment, the seed-origin bacterialendophyte produces a compound with antifungal properties, for example,fungicidal or fungistatic activity against S. cerevisiae and/orRhizoctonia.

In some embodiments, the seed-origin bacterial endophyte is capable ofnitrogen fixation, and is thus capable of producing ammonium fromatmospheric nitrogen. The ability of bacteria to fix nitrogen can beconfirmed by testing for growth of the bacteria in nitrogen-free growthmedia, for example, LGI media, as described herein.

In some embodiments, the seed origin bacterial endophyte can produce acompound which increases the solubility of mineral phosphate in themedium, i.e., mineral phosphate solubilization, for example, using thegrowth assays described herein. In one embodiment, the seed-originbacterial endophyte n produces a compound which allows the bacterium togrow in growth media containing Ca₃HPO₄ as the sole phosphate source.

In some embodiments, the seed-origin bacterial endophyte can produce asiderophore. Siderophores are small high-affinity iron chelating agentssecreted by microorganisms that increase the bioavailability of iron.Siderophore production by the bacterial endophyte can be detected, forexample, using the methods described herein, as well as elsewhere(Perez-Miranda et al., 2007, J Microbiol Methods. 70:127-31,incorporated herein by reference in its entirety).

In some embodiments, the seed-origin bacterial endophyte can produce ahydrolytic enzyme. For example, in one embodiment, a bacterial endophytecan produce a hydrolytic enzyme selected from the group consisting of acellulase, a pectinase, a chitinase and a xylanase. Hydrolytic enzymescan be detectedusing the methods described herein (see also, cellulase:Quadt-Hallmann et al., (1997) Can. J. Microbiol., 43: 577-582;pectinase: Soares et al. (1999). Revista de Microbiolgia 30(4): 299-303;chitinase: Li et al., (2004) Mycologia 96: 526-536; and xylanase: Sutoet al., (2002) J Biosci Bioeng. 93:88-90, each of which is incorporatedby reference in its entirety).

In some embodiment, purified bacterial populations contain synergisticendophytic populations, e.g., synergistic seed-origin bacterialendophytes. As used herein, synergistic endophytic populations refer totwo or more endophyte populations that produce one or more effects(e.g., two or more or three or more effects) that are greater than thesum of their individual effects. For example, in some embodiments, apurified bacterial population contains two or more different seed-originbacterial endophytes that are capable of synergistically increasing atleast one of e.g., production of a phytohormone such as auxin,production of acetoin, production of an antimicrobial compound,production of a siderophore, production of a cellulase, production of apectinase, production of a chitinase, production of a xylanase, nitrogenfixation, or mineral phosphate solubilization in an agricultural grassplant. Synergistically increasing one or more of such properties canincrease a beneficial trait in an agricultural grass plant, such as anincrease in drought tolerance.

In some embodiments, a purified bacterial population containing one ormore seed-origin bacterial endophytes can increase one or moreproperties such as production of a phytohormone such as auxin,production of acetoin, production of an antimicrobial compound,production of a siderophore, production of a cellulase, production of apectinase, production of a chitinase, production of a xylanase, ormineral phosphate solubilization in an agricultural grass plant.,without increasing nitrogen fixation in the agricultural grass plant.

In some embodiments, metabolites in grass plants can be modulated bymaking synthetic combinations of purified bacterial populationscontaining endophytic microbes such as seed-origin bacterial endophytesand a seed or seedling of an agricultural grass plant. For example, abacterial endophyte described herein can cause a detectable modulation(e.g., an increase or decrease) in the level of various metabolites,e.g., indole-3-carboxylic acid, trans-zeatin, abscisic acid, phaseicacid, indole-3-acetic acid, indole-3-butyric acid, indole-3-acrylicacid, jasmonic acid, jasmonic acid methyl ester, dihydrophaseic acid,gibberellin A3, salicylic acid, upon colonization of a grass plant.

In some embodiments, the endophytic microbe modulates the level of themetabolite directly (e.g., the microbe itself produces the metabolite,resulting in an overall increase in the level of the metabolite found inthe plant). In other cases, the agricultural grass plant, as a result ofthe association with the endophytic microbe (e.g., a seed-originbacterial endophyte), exhibits a modulated level of the metabolite(e.g., the plant reduces the expression of a biosynthetic enzymeresponsible for production of the metabolite as a result of the microbeinoculation). In still other cases, the modulation in the level of themetabolite is a consequence of the activity of both the microbe and theplant (e.g., the plant produces increased amounts of the metabolite whencompared with a reference agricultural plant, and the endophytic microbealso produces the metabolite). Therefore, as used herein, a modulationin the level of a metabolite can be an alteration in the metabolitelevel through the actions of the microbe and/or the inoculated plant.

The levels of a metabolite can be measured in an agricultural plant, andcompared with the levels of the metabolite in a reference agriculturalplant, and grown under the same conditions as the inoculated plant. Theuninoculated plant that is used as a reference agricultural plant is aplant which has not been applied with a formulation with the endophyticmicrobe (e.g., a formulation comprising a population of purifiedbacterial endophytes). The uninoculated plant used as the referenceagricultural plant is generally the same species and cultivar as, and isisogenic to, the inoculated plant.

The metabolite whose levels are modulated (e.g., increased or decreased)in the endophyte-associated plant may serve as a primary nutrient (i.e.,it provides nutrition for the humans and/or animals who consume theplant, plant tissue, or the commodity plant product derived therefrom,including, but not limited to, a sugar, a starch, a carbohydrate, aprotein, an oil, a fatty acid, or a vitamin). The metabolite can be acompound that is important for plant growth, development or homeostasis(for example, a phytohormone such as an auxin, cytokinin, gibberellin, abrassinosteroid, ethylene, or abscisic acid, a signaling molecule, or anantioxidant). In other embodiments, the metabolite can have otherfunctions. For example, in one embodiment, a metabolite can havebacteriostatic, bactericidal, fungistatic, fungicidal or antiviralproperties. In other embodiments, the metabolite can haveinsect-repelling, insecticidal, nematode-repelling, or nematicidalproperties. In still other embodiments, the metabolite can serve a rolein protecting the plant from stresses, may help improve plant vigor orthe general health of the plant. In yet another embodiment, themetabolite can be a useful compound for industrial production. Forexample, the metabolite may itself be a useful compound that isextracted for industrial use, or serve as an intermediate for thesynthesis of other compounds used in industry. A level of a metabolitecan be increased by 1%, for example, at least 10%, for example, at least20%, at least 30%, at least 40%, at least 50%, at least 75%, at least100%, at least 150%, at least 200%, at least 300% or more, when comparedwith a reference agricultural plant. In a particular embodiment, thelevel of the metabolite is increased within the agricultural plant or aportion thereof such that it is present at a concentration of at least0.1 μg/g dry weight, for example, at least 0.3 μg/g dry weight, 1.0 μg/gdry weight, 3.0 μg/g dry weight, 10 μg/g dry weight, 30 μg/g dry weight,100 μg/g dry weight, 300 μg/g dry weight, 1 mg/g dry weight, 3 mg/g dryweight, 10 mg/g dry weight, 30 mg/g dry weight, 100 mg/g dry weight ormore, of the plant or portion thereof.

Likewise, the modulation can be a decrease in the level of a metabolite.The reduction can be in a metabolite affecting the taste of a plant or acommodity plant product derived from a plant (for example, a bittertasting compound), or in a metabolite which makes a plant or theresulting commodity plant product otherwise less valuable (for example,reduction of oxalate content in certain plants, or compounds which aredeleterious to human and/or animal health). The metabolite whose levelis to be reduced can be a compound which affects quality of a commodityplant product (e.g., reduction of lignin levels). The level ofmetabolite in the agricultural grass plant or portion thereof can be,for example, decreased by at least 1%, for example, at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 99% or more, when compared with a reference agricultural plant ina reference environment.

In some embodiments, the seed-origin bacterial endophyte is capable ofgenerating a bacterial network in the agricultural grass plant orsurrounding environment of the plant, which network is capable ofcausing a detectable modulation in the level of a metabolite in the hostplant.

In a particular embodiment, the metabolite can serve as a signaling orregulatory molecule. The signaling pathway can be associated with aresponse to a stress, for example, one of the stress conditions selectedfrom the group consisting of drought stress, salt stress, heat stress,cold stress, low nutrient stress, nematode stress, insect herbivorystress, fungal pathogen stress, bacterial pathogen stress, and viralpathogen stress. The inoculated agricultural plant is grown underconditions such that the level of one or more metabolites is modulatedin the plant, wherein the modulation is indicative of increasedresistance to a stress selected from the group consisting of droughtstress, salt stress, heat stress, cold stress, low nutrient stress,nematode stress, insect herbivory stress, fungal pathogen stress,bacterial pathogen stress, and viral pathogen stress. The increasedresistance can be measured at about 10 minutes after applying thestress, for example about 20 minutes, 30 minutes, about 45 minutes,about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12hours, about 16 hours, about 20 hours, about 24 hours, about 36 hours,about 48 hours, about 72 hours, about 96 hours, about 120 hours, orabout a week after applying the stress.

The metabolites or other compounds described herein can be detectedusing any suitable method including, but not limited to gelelectrophoresis, liquid and gas phase chromatography, either alone orcoupled to mass spectrometry (See, for example, the Examples sectionsbelow), NMR (See e.g., U.S. patent publication 20070055456, which isincorporated herein by reference in its entirety), immunoassays(enzyme-linked immunosorbent assays (ELISA)), chemical assays,spectroscopy and the like. In some embodiments, commercial systems forchromatography and NMR analysis are utilized.

In other embodiments, metabolites or other compounds are detected usingoptical imaging techniques such as magnetic resonance spectroscopy(MRS), magnetic resonance imaging (MRI), CAT scans, ultra sound,MS-based tissue imaging or X-ray detection methods (e.g., energydispersive x-ray fluorescence detection).

Any suitable method may be used to analyze the biological sample (e.g.,seed or plant tissue) in order to determine the presence, absence orlevel(s) of the one or more metabolites or other compounds in thesample. Suitable methods include chromatography (e.g., HPLC, gaschromatography, liquid chromatography), mass spectrometry (e.g., MS,MS-MS), LC-MS, enzyme-linked immunosorbent assay (ELISA), antibodylinkage, other immunochemical techniques, biochemical or enzymaticreactions or assays, and combinations thereof. The levels of one or moreof the recited metabolites or compounds may be determined in the methodsof the present invention. For example, the level(s) of one metabolitesor compounds, two or more metabolites, three or more metabolites, fouror more metabolites, five or more metabolites, six or more metabolites,seven or more metabolites, eight or more metabolites, nine or moremetabolites, ten or more metabolites, or compounds etc., including acombination of some or all of the metabolites or compounds including,but not limited to those disclosed herein may be determined and used insuch methods.

As shown in the Examples and otherwise herein, endophyte-inoculatedplants display increased thermal tolerance, herbicide tolerance, droughtresistance, insect resistance, fungus resistance, virus resistance,bacteria resistance, male sterility, cold tolerance, salt tolerance,increased yield, enhanced nutrient use efficiency, increased nitrogenuse efficiency, increased protein content, increased fermentablecarbohydrate content, reduced lignin content, increased antioxidantcontent, enhanced water use efficiency, increased vigor, increasedgermination efficiency, earlier or increased flowering, increasedbiomass, altered root-to-shoot biomass ratio, enhanced soil waterretention, or a combination thereof. Therefore, in one embodiment, thebacterial endophytic population is disposed on the surface or within atissue of the seed or seedling in an amount effective to increase thebiomass of the plant, or a part or tissue of the plant grown from theseed or seedling. The increased biomass is useful in the production ofcommodity products derived from the plant. Such commodity productsinclude an animal feed, a fish fodder, a cereal product, a processedhuman-food product, a sugar or an alcohol. Such products may be afermentation product or a fermentable product, one such exemplaryproduct is a biofuel. The increase in biomass can occur in a part of theplant (e.g., the root tissue, shoots, leaves, etc.), or can be anincrease in overall biomass. Increased biomass production, such anincrease meaning at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, or greater than 100% when compared with a referenceagricultural plant. Such increase in overall biomass can be underrelatively stress-free conditions. In other cases, the increase inbiomass can be in plants grown under any number of abiotic or bioticstresses, including drought stress, salt stress, heat stress, coldstress, low nutrient stress, nematode stress, insect herbivory stress,fungal pathogen stress, bacterial pathogen stress, and viral pathogenstress. In one particular embodiment, the bacterial endophyticpopulation is disposed in an amount effective to increase root biomassby at least 10%, for example, at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 75%, at least 100%, or more, whencompared with a reference agricultural plant.

In another embodiment, the bacterial endophytic population is disposedon the surface or within a tissue of the seed or seedling in an amounteffective to increase the rate of seed germination when compared with areference agricultural plant. For example, the increase in seedgermination can be at least 10%, for example, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 75%, at least100%, or more, when compared with a reference agricultural plant.

In other cases, the endophytic microbe is disposed on the seed orseedling in an amount effective to increase the average biomass of thefruit or cob from the resulting plant by at least 10%, for example, atleast 20%, at least 30%, at least 40%, at least 50%, at least 75%, atleast 100% or more, when compared with a reference agricultural plant.

As highlighted in the Examples section, plants inoculated with abacterial endophytic population also show an increase in overall plantheight. Therefore, in one embodiment, the present invention provides fora seed comprising a bacterial endophytic population which is disposed onthe surface or within a tissue of the seed or seedling in an amounteffective to increase the height of the plant. For example, thebacterial endophytic population is disposed in an amount effective toresult in an increase in height of the agricultural plant such that isat least 10% greater, for example, at least 20% greater, at least 30%greater, at least 40% greater, at least 50% greater, at least 60%greater, at least 70% greater, at least 80% greater, at least 90%greater, at least 100% greater, at least 125% greater, at least 150%greater or more, when compared with a reference agricultural plant. Suchan increase in height can be under relatively stress-free conditions. Inother cases, the increase in height can be in plants grown under anynumber of abiotic or biotic stresses, including drought stress, saltstress, heat stress, cold stress, low nutrient stress, nematode stress,insect herbivory stress, fungal pathogen stress, bacterial pathogenstress, or viral pathogen stress.

The host plants inoculated with the bacterial endophytic population alsoshow dramatic improvements in their ability to utilize water moreefficiently. Water use efficiency is a parameter often correlated withdrought tolerance. Water use efficiency (WUE) is a parameter oftencorrelated with drought tolerance, and is the CO2 assimilation rate perwater transpired by the plant. An increase in biomass at low wateravailability may be due to relatively improved efficiency of growth orreduced water consumption. In selecting traits for improving crops, adecrease in water use, without a change in growth would have particularmerit in an irrigated agricultural system where the water input costswere high. An increase in growth without a corresponding jump in wateruse would have applicability to all agricultural systems. In manyagricultural systems where water supply is not limiting, an increase ingrowth, even if it came at the expense of an increase in water use alsoincreases yield.

When soil water is depleted or if water is not available during periodsof drought, crop yields are restricted. Plant water deficit develops iftranspiration from leaves exceeds the supply of water from the roots.The available water supply is related to the amount of water held in thesoil and the ability of the plant to reach that water with its rootsystem. Transpiration of water from leaves is linked to the fixation ofcarbon dioxide by photosynthesis through the stomata. The two processesare positively correlated so that high carbon dioxide influx throughphotosynthesis is closely linked to water loss by transpiration. Aswater transpires from the leaf, leaf water potential is reduced and thestomata tend to close in a hydraulic process limiting the amount ofphotosynthesis. Since crop yield is dependent on the fixation of carbondioxide in photosynthesis, water uptake and transpiration arecontributing factors to crop yield. Plants which are able to use lesswater to fix the same amount of carbon dioxide or which are able tofunction normally at a lower water potential have the potential toconduct more photosynthesis and thereby to produce more biomass andeconomic yield in many agricultural systems. An increased water useefficiency of the plant relates in some cases to an increasedfruit/kernel size or number.

Therefore, in one embodiment, the plants described herein exhibit anincreased water use efficiency (WUE) when compared with a referenceagricultural plant grown under the same conditions. For example, theplants grown from the seeds comprising the bacterial endophyticpopulation can have at least 5% higher WUE, for example, at least 10%higher, at least 20% higher, at least 30% higher, at least 40% higher,at least 50% higher, at least 60% higher, at least 70% higher, at least80% higher, at least 90% higher, at least 100% higher WUE than areference agricultural plant grown under the same conditions. Such anincrease in WUE can occur under conditions without water deficit, orunder conditions of water deficit, for example, when the soil watercontent is less than or equal to 60% of water saturated soil, forexample, less than or equal to 50%, less than or equal to 40%, less thanor equal to 30%, less than or equal to 20%, less than or equal to 10% ofwater saturated soil on a weight basis.

In a related embodiment, the plant comprising the bacterial endophytecan have at least 10% higher relative water content (RWC), for example,at least 20% higher, at least 30% higher, at least 40% higher, at least50% higher, at least 60% higher, at least 70% higher, at least 80%higher, at least 90% higher, at least 100% higher RWC than a referenceagricultural plant grown under the same conditions.

Synthetic Combinations and Methods of Making

As shown in the Examples section below, the bacterial endophyticpopulations described herein are capable of colonizing a host plant.Successful colonization can be confirmed by detecting the presence ofthe bacterial population within the plant. For example, after applyingthe bacteria to the seeds, high titers of the bacteria can be detectedin the roots and shoots of the plants that germinate from the seeds. Inaddition, significant quantities of the bacteria can be detected in therhizosphere of the plants. Detecting the presence of the endophyticmicrobe inside the plant can be accomplished by measuring the viabilityof the microbe after surface sterilization of the seed or the plant:endophytic colonization results in an internal localization of themicrobe, rendering it resistant to conditions of surface sterilization.The presence and quantity of the microbe can also be established usingother means known in the art, for example, immunofluorescence microscopyusing microbe specific antibodies, or fluorescence in situ hybridization(see, for example, Amann et al. (2001) Current Opinion in Biotechnology12:231-236, incorporated herein by reference in its entirety).Alternatively, specific nucleic acid probes recognizing conservedsequences from the endophytic bacterium can be employed to amplify aregion, for example by quantitative PCR, and correlated to CFUs by meansof a standard curve.

In another embodiment, the endophytic microbe is disposed, for example,on the surface of a seed of an agricultural grass plant, in an amounteffective to be detectable in the mature agricultural plant. In oneembodiment, the endophytic microbe is disposed in an amount effective tobe detectable in an amount of at least about 100 CFU, at least about 200CFU, at least about 300 CFU, at least about 500 CFU, at least about1,000 CFU, at least about 3,000 CFU, at least about 10,000 CFU, at leastabout 30,000 CFU, at least about 100,000 CFU or more in the matureagricultural plant.

In some cases, the endophytic microbe is capable of colonizingparticular tissue types of the plant. In one embodiment, the endophyticmicrobe is disposed on the seed or seedling in an amount effective to bedetectable within a target tissue of the mature agricultural plantselected from a fruit, a seed, a leaf, or a root, or portion thereof.For example, the endophytic microbe can be detected in an amount of atleast about 100 CFU, at least about 200 CFU, at least about 300 CFU, atleast about 500 CFU, at least about 1,000 CFU, at least about 3,000 CFU,at least about 10,000 CFU, at least about 30,000 CFU, at least about100,000 CFU or more, in the target tissue of the mature agriculturalplant.

Endophytes Compatible with Agrichemicals.

In certain embodiments, the endophyte is selected on the basis of itscompatibility with commonly used agrichemicals. As mentioned earlier,plants, particularly agricultural plants, can be treated with a vastarray of agrichemicals, including fungicides, biocides (anti-bacterialagents), herbicides, insecticides, nematicides, rodenticides,fertilizers, and other agents.

In some cases, it can be important for the endophyte to be compatiblewith agrichemicals, particularly those with fungicidal or antibacterialproperties, in order to persist in the plant although, as mentionedearlier, there are many such fungicidal or antibacterial agents that donot penetrate the plant, at least at a concentration sufficient tointerfere with the endophyte. Therefore, where a systemic fungicide orantibacterial agent is used in the plant, compatibility of the endophyteto be inoculated with such agents will be an important criterion.

In one embodiment, natural isolates of endophytes which are compatiblewith agrichemicals can be used to inoculate the plants according to themethods described herein. For example, fungal endophytes which arecompatible with agriculturally employed fungicides can be isolated byplating a culture of the endophytes on a petri dish containing aneffective concentration of the fungicide, and isolating colonies of theendophyte that are compatible with the fungicide. In another embodiment,an endophyte that is compatible with a fungicide is used for the methodsdescribed herein. For example, the endophyte can be compatible with atleast one of the fungicides selected from the group consisting of:2-(thiocyanatomethylthio)-benzothiazole, 2-phenylphenol,8-hydroxyquinoline sulfate, ametoctradin, amisulbrom, antimycin,Ampelomyces quisqualis, azaconazole, azoxystrobin, Bacillus subtilis,benalaxyl, benomyl, benthiavalicarb-isopropyl,benzylaminobenzene-sulfonate (BAB S) salt, bicarbonates, biphenyl,bismerthiazol, bitertanol, bixafen, blasticidin-S, borax, Bordeauxmixture, boscalid, bromuconazole, bupirimate, calcium polysulfide,captafol, captan, carbendazim, carboxin, carpropamid, carvone,chloroneb, chlorothalonil, chlozolinate, Coniothyrium minitans, copperhydroxide, copper octanoate, copper oxychloride, copper sulfate, coppersulfate (tribasic), cuprous oxide, cyazofamid, cyflufenamid, cymoxanil,cyproconazole, cyprodinil, dazomet, debacarb, diammoniumethylenebis-(dithiocarbamate), dichlofluanid, dichlorophen, diclocymet,diclomezine, dichloran, diethofencarb, difenoconazole, difenzoquat ion,diflumetorim, dimethomorph, dimoxystrobin, diniconazole, diniconazole-M,dinobuton, dinocap, diphenylamine, dithianon, dodemorph, dodemorphacetate, dodine, dodine free base, edifenphos, enestrobin,epoxiconazole, ethaboxam, ethoxyquin, etridiazole, famoxadone,fenamidone, fenarimol, fenbuconazole, fenfuram, fenhexamid, fenoxanil,fenpiclonil, fenpropidin, fenpropimorph, fentin, fentin acetate, fentinhydroxide, ferbam, ferimzone, fluazinam, fludioxonil, flumorph,fluopicolide, fluopyram, fluoroimide, fluoxastrobin, fluquinconazole,flusilazole, flusulfamide, flutianil, flutolanil, flutriafol,fluxapyroxad, folpet, formaldehyde, fosetyl, fosetyl-aluminium,fuberidazole, furalaxyl, furametpyr, guazatine, guazatine acetates,GY-81, hexachlorobenzene, hexaconazole, hymexazol, imazalil, imazalilsulfate, imibenconazole, iminoctadine, iminoctadine triacetate,iminoctadine tris(albesilate), ipconazole, iprobenfos, iprodione,iprovalicarb, isoprothiolane, isopyrazam, isotianil, kasugamycin,kasugamycin hydrochloride hydrate, kresoxim-methyl, mancopper, mancozeb,mandipropamid, maneb, mepanipyrim, mepronil, mercuric chloride, mercuricoxide, mercurous chloride, metalaxyl, mefenoxam, metalaxyl-M, metam,metam-ammonium, metam-potassium, metam-sodium, metconazole,methasulfocarb, methyl iodide, methyl isothiocyanate, metiram,metominostrobin, metrafenone, mildiomycin, myclobutanil, nabam,nitrothal-isopropyl, nuarimol, octhilinone, ofurace, oleic acid (fattyacids), orysastrobin, oxadixyl, oxine-copper, oxpoconazole fumarate,oxycarboxin, pefurazoate, penconazole, pencycuron, penflufen,pentachlorophenol, pentachlorophenyl laurate, penthiopyrad,phenylmercury acetate, phosphonic acid, phthalide, picoxystrobin,polyoxin B, polyoxins, polyoxorim, potassium bicarbonate, potassiumhydroxyquinoline sulfate, probenazole, prochloraz, procymidone,propamocarb, propamocarb hydrochloride, propiconazole, propineb,proquinazid, prothioconazole, pyraclostrobin, pyrametostrobin,pyraoxystrobin, pyrazophos, pyribencarb, pyributicarb, pyrifenox,pyrimethanil, pyroquilon, quinoclamine, quinoxyfen, quintozene,Reynoutria sachalinensis extract, sedaxane, silthiofam, simeconazole,sodium 2-phenylphenoxide, sodium bicarbonate, sodiumpentachlorophenoxide, spiroxamine, sulfur, SYP-Z071, SYP-Z048, tar oils,tebuconazole, tebufloquin, tecnazene, tetraconazole, thiabendazole,thifluzamide, thiophanate-methyl, thiram, tiadinil, tolclofos-methyl,tolylfluanid, triadimefon, triadimenol, triazoxide, tricyclazole,tridemorph, trifloxystrobin, triflumizole, triforine, triticonazole,validamycin, valifenalate, valiphenal, vinclozolin, zineb, ziram,zoxamide, Candida oleophila, Fusarium oxysporum, Gliocladium spp.,Phlebiopsis gigantea, Streptomyces griseoviridis, Trichoderma spp.,(RS)—N-(3,5-dichlorophenyl)-2-(methoxymethyl)-succinimide,1,2-dichloropropane, 1,3-dichloro-1,1,3,3-tetrafluoroacetone hydrate,1-chloro-2,4-dinitronaphthalene, 1-chloro-2-nitropropane,2-(2-heptadecyl-2-imidazolin-1-yl)ethanol,2,3-dihydro-5-phenyl-1,4-dithi-ine 1,1,4,4-tetraoxide,2-methoxyethylmercury acetate, 2-methoxyethylmercury chloride,2-methoxyethylmercury silicate, 3-(4-chlorophenyl)-5-methylrhodanine,4-(2-nitroprop-1-enyl)phenyl thiocyanateme, ampropylfos, anilazine,azithiram, barium polysulfide, Bayer 32394, benodanil, benquinox,bentaluron, benzamacril; benzamacril-isobutyl, benzamorf, binapacryl,bis(methylmercury) sulfate, bis(tributyltin) oxide, buthiobate, cadmiumcalcium copper zinc chromate sulfate, carbamorph, CECA, chlobenthiazone,chloraniformethan, chlorfenazole, chlorquinox, climbazole, cyclafuramid,cypendazole, cyprofuram, decafentin, dichlone, dichlozoline,diclobutrazol, dimethirimol, dinocton, dinosulfon, dinoterbon,dipyrithione, ditalimfos, dodicin, drazoxolon, EBP, ESBP, etaconazole,etem, ethirim, fenaminosulf, fenapanil, fenitropan, 5-fluorocytosine andprofungicides thereof, fluotrimazole, furcarbanil, furconazole,furconazole-cis, furmecyclox, furophanate, glyodine, griseofulvin,halacrinate, Hercules 3944, hexylthiofos, ICIA0858, isopamphos,isovaledione, mebenil, mecarbinzid, metazoxolon, methfuroxam,methylmercury dicyandiamide, metsulfovax, milneb, mucochloric anhydride,myclozolin, N-3,5-dichlorophenyl-succinimide,N-3-nitrophenylitaconimide, natamycin,N-ethylmercurio-4-toluenesulfonanilide, nickelbis(dimethyldithiocarbamate), OCH, phenylmercurydimethyldithiocarbamate, phenylmercury nitrate, phosdiphen, picolinamideUK-2A and derivatives thereof, prothiocarb; prothiocarb hydrochloride,pyracarbolid, pyridinitril, pyroxychlor, pyroxyfur, quinacetol;quinacetol sulfate, quinazamid, quinconazole, rabenzazole,salicylanilide, SSF-109, sultropen, tecoram, thiadifluor, thicyofen,thiochlorfenphim, thiophanate, thioquinox, tioxymid, triamiphos,triarimol, triazbutil, trichlamide, urbacid, XRD-563, and zarilamide,IK-1140.

In still another embodiment, an endophyte that is compatible with anantibacterial compound is used for the methods described herein. Forexample, the endophyte can be compatible with at least one of theantibiotics selected from the group consisting of: Amikacin, Gentamicin,Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Spectinomycin,Geldanamycin, Herbimycin, Rifaximin, streptomycin, Loracarbef,Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil,Cefazolin, Cefalotin or Cefalothin, Cefalexin, Cefaclor, Cefamandole,Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren,Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten,Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole,Teicoplanin, Vancomycin, Telavancin, Clindamycin, Lincomycin,Daptomycin, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin,Roxithromycin, Troleandomycin, Telithromycin, Spiramycin, Aztreonam,Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid,Torezolid, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin,Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin,Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin,Penicillin G, Temocillin, Ticarcillin, Amoxicillin/clavulanate,Ampicillin/sulbactam, Piperacillin/tazobactam, Ticarcillin/clavulanate,Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin,Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid,Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin,Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silversulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole,Sulfanilimide (archaic), Sulfasalazine, Sulfisoxazole,Trimethoprim-Sulfamethoxazole (Co-trimoxazole) (TMP-SMX),Sulfonamidochrysoidine (archaic), Demeclocycline, Doxycycline,Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone,Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid,Pyrazinamide, Rifampicin (Rifampin in US), Rifabutin, Rifapentine,Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid,Metronidazole, Mupirocin, Platensimycin, Quinupristin/Dalfopristin,Thiamphenicol, Tigecycline, Tinidazole, and Trimethoprim. Fungicidecompatible endophytes can also be isolated by selection on liquidmedium. The culture of endophytes can be plated on petri dishes withoutany forms of mutagenesis; alternatively, the endophytes can bemutagenized using any means known in the art. For example, microbialcultures can be exposed to UV light, gamma-irradiation, or chemicalmutagens such as ethylmethanesulfonate (EMS) prior to selection onfungicide containing media. Finally, where the mechanism of action of aparticular fungicide is known, the target gene can be specificallymutated (either by gene deletion, gene replacement, site-directedmutagenesis, etc.) to generate an endophyte that is resilient againstthat particular fungicide. It is noted that the above-described methodscan be used to isolate fungi that are compatible with both fungistaticand fungicidal compounds.

It will also be appreciated by one skilled in the art that a plant maybe exposed to multiple types of fungicides or antibacterial compounds,either simultaneously or in succession, for example at different stagesof plant growth. Where the target plant is likely to be exposed tomultiple fungicidal and/or antibacterial agents, an endophyte that iscompatible with many or all of these agrichemicals can be used toinoculate the plant. An endophyte that is compatible with severalfungicidal agents can be isolated, for example, by serial selection. Anendophyte that is compatible with the first fungicidal agent is isolatedas described above (with or without prior mutagenesis). A culture of theresulting endophyte can then be selected for the ability to grow onliquid or solid media containing the second antifungal compound (again,with or without prior mutagenesis). Colonies isolated from the secondselection are then tested to confirm its compatibility to bothantifungal compounds.

Likewise, bacterial endophytes that are compatible to biocides(including herbicides such as glyphosate or antibacterial compounds,whether bacteriostatic or bactericidal) that are agriculturally employedcan be isolated using methods similar to those described for isolatingfungicide compatible endophytes. In one embodiment, mutagenesis of themicrobial population can be performed prior to selection with anantibacterial agent. In another embodiment, selection is performed onthe microbial population without prior mutagenesis. In still anotherembodiment, serial selection is performed on an endophyte: the endophyteis first selected for compatibility to a first antibacterial agent. Theisolated compatible endophyte is then cultured and selected forcompatibility to the second antibacterial agent. Any colony thusisolated is tested for compatibility to each, or both antibacterialagents to confirm compatibility with these two agents.

Compatibility with an antimicrobial agent can be determined by a numberof means known in the art, including the comparison of the minimalinhibitory concentration (MIC) of the unmodified and modified endophyte.Therefore, in one embodiment, the present invention discloses anisolated modified endophyte derived from an endophyte isolated fromwithin a plant or tissue thereof, wherein the endophyte is modified suchthat it exhibits at least 3 fold greater, for example, at least 5 foldgreater, at least 10 fold greater, at least 20 fold greater, at least 30fold greater or more MIC to an antimicrobial agent when compared withthe unmodified endophyte.

In one particular aspect, disclosed herein are bacterial endophytes withenhanced compatibility to the herbicide glyphosate. In one embodiment,the bacterial endophyte has a doubling time in growth medium containingat least 1 mM glyphosate, for example, at least 2 mM glyphosate, atleast 5 mM glyphosate, at least 10 mM glyphosate, at least 15 mMglyphosate or more, that is no more than 250%, for example, no more than200%, no more than 175%, no more than 150%, or no more than 125%, of thedoubling time of the endophyte in the same growth medium containing noglyphosate. In one particular embodiment, the bacterial endophyte has adoubling time in growth medium containing 5 mM glyphosate that is nomore than 150% the doubling time of the endophyte in the same growthmedium containing no glyphosate.

In another embodiment, the bacterial endophyte has a doubling time in aplant tissue containing at least 10 ppm glyphosate, for example, atleast 15 ppm glyphosate, at least 20 ppm glyphosate, at least 30 ppmglyphosate, at least 40 ppm glyphosate or more, that is no more than250%, for example, no more than 200%, no more than 175%, no more than150%, or no more than 125%, of the doubling time of the endophyte in areference plant tissue containing no glyphosate. In one particularembodiment, the bacterial endophyte has a doubling time in a planttissue containing 40 ppm glyphosate that is no more than 150% thedoubling time of the endophyte in a reference plant tissue containing noglyphosate.

The selection process described above can be repeated to identifyisolates of the endophyte that are compatible with a multitude ofantifungal or antibacterial agents.

Candidate isolates can be tested to ensure that the selection foragrichemical compatibility did not result in loss of a desired microbialbioactivity. Isolates of the endophyte that are compatible with commonlyemployed fungicides can be selected as described above. The resultingcompatible endophyte can be compared with the parental endophyte onplants in its ability to promote germination.

The agrichemical compatible endophytes generated as described above canbe detected in samples. For example, where a transgene was introduced torender the endophyte compatible with the agrichemical(s), the transgenecan be used as a target gene for amplification and detection by PCR. Inaddition, where point mutations or deletions to a portion of a specificgene or a number of genes results in compatibility with theagrichemical(s), the unique point mutations can likewise be detected byPCR or other means known in the art. Such methods allow the detection ofthe microbe even if it is no longer viable. Thus, commodity plantproducts produced using the agrichemical compatible microbes describedherein can readily be identified by employing these and related methodsof nucleic acid detection.

Beneficial Attributes of Synthetic Combinations of Cereal Seeds andSeed-Origin Endophytes

Improved Attributes Conferred by the Endophyte.

The present invention contemplates the establishment of a microbialsymbiont in a plant. In one embodiment, the microbial associationresults in a detectable change to the seed or plant. The detectablechange can be an improvement in a number of agronomic traits (e.g.,improved general health, increased response to biotic or abioticstresses, or enhanced properties of the plant or a plant part, includingfruits and grains). Alternatively, the detectable change can be aphysiological or biological change that can be measured by methods knownin the art. The detectable changes are described in more detail in thesections below. As used herein, an endophyte is considered to haveconferred an improved agricultural trait whether or not the improvedtrait arose from the plant, the endophyte, or the concerted actionbetween the plant and endophyte. Therefore, for example, whether abeneficial hormone or chemical is produced by the plant or endophyte,for purposes of the present invention, the endophyte will be consideredto have conferred an improved agronomic trait upon the host plant.

In some aspects, provided herein, are methods for producing a seed of aplant with a heritably altered trait. The trait of the plant can bealtered without known genetic modification of the plant genome, andcomprises the following steps. First, a preparation of an isolatedendophyte which is exogenous to the seed of the plant is provided, andoptionally processed to produce a microbial preparation. The microbialpreparation is then contacted with the plant. The plants are thenallowed to go to seed, and the seeds, which contain the endophytes onand/or in the seed are collected. The endophytes contained within theseed are viably incorporated into the seed.

The method of the present invention can facilitate crop productivity byenhancing germination, seedling vigor and biomass in comparison with anon-treated control. Moreover, the introduction of the beneficialmicroorganisms to within the seed instead of by, e.g., seed coating,makes the endophytes less susceptible to environmental perturbation andmore compatible with chemical seed coatings (e.g., pesticides andherbicides). Using endophyte colonized seeds, the plant growth andbiomass increases are statistically similar to those obtained usingconventional inoculation methods e.g., exogenous seed soaking and soilinoculation (that are more laborious and less practicable in certaincircumstances).

Improved General Health.

Also described herein are plants, and fields of plants, that areassociated with beneficial bacterial and/or fungal endophytes, such thatthe overall fitness, productivity or health of the plant or a portionthereof, is maintained, increased and/or improved over a period of time.Improvement in overall plant health can be assessed using numerousphysiological parameters including, but not limited to, height, overallbiomass, root and/or shoot biomass, seed germination, seedling survival,photosynthetic efficiency, transpiration rate, seed/fruit number ormass, plant grain or fruit yield, leaf chlorophyll content,photosynthetic rate, root length, or any combination thereof. Improvedplant health, or improved field health, can also be demonstrated throughimproved resistance or response to a given stress, either biotic orabiotic stress, or a combination of one or more abiotic stresses, asprovided herein.

Other Abiotic Stresses.

Disclosed herein are endophyte-associated plants with increasedresistance to an abiotic stress. Exemplary abiotic stresses include, butare not limited to:

Drought and Heat Tolerance.

In some cases, a plant resulting from seeds containing the endophyte canexhibit a physiological change, such as a decreased change inphotosynthetic activity (expressed, for example, as ΔFv/Fm) afterexposure to heat shock or drought conditions as compared to acorresponding control, genetically identical plant that does not containthe endophytes grown in the same conditions. In some cases, theendophyte-associated plant as disclosed herein can exhibit an increasedchange in photosynthetic activity ΔFv(ΔFv/Fm) after heat-shock ordrought stress treatment, for example 1, 2, 3, 4, 5, 6, 7 days or moreafter the heat-shock or drought stress treatment, or untilphotosynthesis ceases, as compared with corresponding control plant ofsimilar developmental stage but not containing the endophytes. Forexample, a plant having an endophyte able to confer heat and/ordrought-tolerance can exhibit a ΔFv/Fm of from about 0.1 to about 0.8after exposure to heat-shock or drought stress or a ΔFv/Fm range of fromabout 0.03 to about 0.8 under one day, or 1, 2, 3, 4, 5, 6, 7, or over 7days post heat-shock or drought stress treatment, or untilphotosynthesis ceases. In some embodiments, stress-induced reductions inphotosynthetic activity can be reduced by at least about 0.25% (forexample, at least about 0.5%, at least about 1%, at least about 2%, atleast about 3, at least about 5%, at least about 8%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 75%, at least about 80%, at least about 80%, at leastabout 90%, at least about 95%, at least about 99% or at least 100%) ascompared to the photosynthetic activity decrease in a correspondingreference agricultural plant following heat shock conditions.Significance of the difference between the endophyte-associated andreference agricultural plants can be established upon demonstratingstatistical significance, for example at p<0.05 with an appropriateparametric or non-parametric statistic, e.g., Chi-square test, Student'st-test, Mann-Whitney test, or F-test based on the assumption or knownfacts that the endophyte-associated plant and reference agriculturalplant have identical or near identical genomes.

In some embodiments, the plants contain endophytes able to confer novelheat and/or drought-tolerance in sufficient quantity, such thatincreased growth under conditions of heat or drought stress is observed.For example, a heat and/or drought-tolerance endophyte populationdescribed herein can be present in sufficient quantity in a plant,resulting in increased growth as compared to a plant that does notcontain the endophyte, when grown under drought conditions or heat shockconditions, or following such conditions. Growth can be assessed withphysiological parameters including, but not limited to, height, overallbiomass, root and/or shoot biomass, seed germination, seedling survival,photosynthetic efficiency, transpiration rate, seed/fruit number ormass, plant grain or fruit yield, leaf chlorophyll content,photosynthetic rate, root length, or any combination thereof.

In some cases, a plant resulting from seeds containing an endophyte thatincludes a novel heat and/or drought tolerance endophyte populationdescribed herein exhibits a difference in the physiological parameterthat is at least about 5% greater, for example at least about 5%, atleast about 8%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 75%, at least about80%, at least about 80%, at least about 90%, or at least 100%, at leastabout 200%, at least about 300%, at least about 400% or greater than areference agricultural plant grown under similar conditions.

In various embodiments, the endophytes introduced into altered seedmicrobiota can confer in the resulting plant thermal tolerance,herbicide tolerance, drought resistance, insect resistance, fungusresistance, virus resistance, bacteria resistance, male sterility, coldtolerance, salt tolerance, increased yield, enhanced nutrient useefficiency, increased nitrogen use efficiency, increased proteincontent, increased fermentable carbohydrate content, reduced lignincontent, increased antioxidant content, enhanced water use efficiency,increased vigor, increased germination efficiency, earlier or increasedflowering, increased biomass, altered root-to-shoot biomass ratio,enhanced soil water retention, or a combination thereof. A differencebetween endophyte-associated plant and a reference agricultural plantcan also be measured using other methods known in the art (see, forexample, Haake et al. (2002) Plant Physiol. 130: 639-648)

Salt Stress.

In other embodiments, endophytes able to confer increased tolerance tosalinity stress can be introduced into plants. The resulting plantscontaining the endophytes can exhibit increased resistance to saltstress, whether measured in terms of survival under saline conditions,or overall growth during, or following salt stress. The physiologicalparameters of plant health recited above, including height, overallbiomass, root and/or shoot biomass, seed germination, seedling survival,photosynthetic efficiency, transpiration rate, seed/fruit number ormass, plant grain or fruit yield, leaf chlorophyll content,photosynthetic rate, root length, or any combination thereof, can beused to measure growth, and compared with the growth rate of referenceagricultural plants (e.g., isogenic plants without the endophytes) grownunder identical conditions. In some cases, a plant resulting from seedscontaining an endophyte able to confer salt tolerance described hereinexhibits a difference in the physiological parameter that is at leastabout 5% greater, for example at least about 5%, at least about 8%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 75%, at least about 80%, at least about80%, at least about 90%, or at least 100%, at least about 200%, at leastabout 300%, at least about 400% or greater than a reference agriculturalplant grown under the same sodium concentration in the soil.

In other instances, endophyte-associated plants and referenceagricultural plants can be grown in soil or growth media containingdifferent concentration of sodium to establish the inhibitoryconcentration of sodium (expressed, for example, as the concentration inwhich growth of the plant is inhibited by 50% when compared with plantsgrown under no sodium stress). Therefore, in another embodiment, a plantresulting from seeds containing an endophyte able to confer salttolerance described herein exhibits an increase in the inhibitory sodiumconcentration by at least 10 mM, for example at least 15 mM, at least 20mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, atleast 70 mM, at least 80 mM, at least 90 mM, at least 100 mM or more,when compared with the reference agricultural plants.

High Metal Content.

Plants are sessile organisms and therefore must contend with theenvironment in which they are placed. While plants have adapted manymechanisms to deal with chemicals and substances that may be deleteriousto their health, heavy metals represent a class of toxins which arehighly relevant for plant growth and agriculture. Plants use a number ofmechanisms to cope with toxic levels of heavy metals (for example,nickel, cadmium, lead, mercury, arsenic, or aluminum) in the soil,including excretion and internal sequestration. For agriculturalpurposes, it is important to have plants that are able to tolerateotherwise hostile conditions, for example soils containing elevatedlevels of toxic heavy metals. Endophytes that are able to conferincreased heavy metal tolerance may do so by enhancing sequestration ofthe metal in certain compartments. Use of such endophytes in a plantwould allow the development of novel plant-endophyte combinations forpurposes of environmental remediation (also known as phytoremediation).Therefore, in one embodiment, the plant containing the endophyte able toconfer increased metal tolerance exhibits a difference in aphysiological parameter that is at least about 5% greater, for exampleat least about 5%, at least about 8%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about75%, at least about 80%, at least about 80%, at least about 90%, or atleast 100%, at least about 200%, at least about 300%, at least about400% or greater than a reference agricultural plant grown under the sameheavy metal concentration in the soil.

Alternatively, the inhibitory concentration of the heavy metal can bedetermined for the endophyte-associated plant and compared with areference agricultural plant under the same conditions. Therefore, inone embodiment, the plants resulting from seeds containing an endophyteable to confer heavy metal tolerance described herein exhibit anincrease in the inhibitory sodium concentration by at least 0.1 mM, forexample at least 0.3 mM, at least 0.5 mM, at least 1 mM, at least 2 mM,at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least30 mM, at least 50 mM or more, when compared with the referenceagricultural plants.

Finally, plants inoculated with endophytes that are able to conferincreased metal tolerance exhibits an increase in overall metalaccumulation by at least 10%, for example at least 15%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 75%, atleast 100%, at least 150%, at least 200%, at least 300% or more, whencompared with uninoculated plants grown under the same conditions.

Low Nutrient Stress.

The endophytes described herein may also confer to the plant anincreased ability to grow in nutrient limiting conditions, for exampleby solubilizing or otherwise making available to the plantsmacronutrients or micronutrients that are complexed, insoluble, orotherwise in an unavailable form. In one embodiment, a plant isinoculated with an endophyte that confers increased ability to liberateand/or otherwise provide to the plant with nutrients selected from thegroup consisting of phosphate, nitrogen, potassium, iron, manganese,calcium, molybdenum, vitamins, or other micronutrients. Such a plant canexhibit increased growth in soil containing limiting amounts of suchnutrients when compared with reference agricultural plant. Differencesbetween the endophyte-associated plant and reference agricultural plantcan be measured by comparing the biomass of the two plant types grownunder limiting conditions, or by measuring the physical parametersdescribed above. Therefore, in one embodiment, the plant containing theendophyte able to confer increased tolerance to nutrient limitingconditions exhibits a difference in a physiological parameter that is atleast about 5% greater, for example at least about 5%, at least about8%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 75%, at least about 80%, at leastabout 80%, at least about 90%, or at least 100%, at least about 200%, atleast about 300%, at least about 400% or greater than a referenceagricultural plant grown under the same heavy metal concentration in thesoil.

Cold Stress.

In some cases, endophytes can confer to the plant the ability totolerate cold stress. Many known methods exist for the measurement of aplant's tolerance to cold stress (as reviewed, for example, in Thomashow(2001) Plant Physiol. 125: 89-93, and Gilmour et al. (2000) PlantPhysiol. 124: 1854-1865, both of which are incorporated herein byreference in their entirety). As used herein, cold stress refers to boththe stress induced by chilling (0° C.-15° C.) and freezing (<0° C.).Some cultivars of agricultural plants can be particularly sensitive tocold stress, but cold tolerance traits may be multigenic, making thebreeding process difficult. Endophytes able to confer cold tolerancewould potentially reduce the damage suffered by farmers on an annualbasis. Improved response to cold stress can be measured by survival ofplants, the amount of necrosis of parts of the plant, or a change incrop yield loss, as well as the physiological parameters used in otherexamples. Therefore, in one embodiment, the plant containing theendophyte able to confer increased cold tolerance exhibits a differencein a physiological parameter that is at least about 5% greater, forexample at least about 5%, at least about 8%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 75%, at least about 80%, at least about 80%, at least about90%, or at least 100%, at least about 200%, at least about 300%, atleast about 400% or greater than a reference agricultural plant grownunder the same conditions of cold stress.

Biotic Stress.

In other embodiments, the bacterial endophyte protects the plant from abiotic stress, for example, insect infestation, nematode infestation,bacterial infection, fungal infection, oomycete infection, protozoalinfection, viral infection, and herbivore grazing, or a combinationthereof.

Insect Herbivory.

There is an abundance of insect pest species that can infect or infest awide variety of plants. Pest infestation can lead to significant damage.Insect pests that infest plant species are particularly problematic inagriculture as they can cause serious damage to crops and significantlyreduce plant yields. A wide variety of different types of plant aresusceptible to pest infestation including commercial crops such ascotton, soybean, wheat, barley, and corn.

In some cases, the endophytes described herein may confer upon the hostplant the ability to repel insect herbivores. In other cases, theendophytes may produce, or induce the production in the plant of,compounds which are insecticidal or insect repellant. The insect may beany one of the common pathogenic insects affecting plants, particularlyagricultural plants. Examples include, but are not limited to:Leptinotarsa spp. (e.g., L. decemlineata (Colorado potato beetle), L.juncta (false potato beetle), or L. texana (Texan false potato beetle));Nilaparvata spp. (e.g., N. lugens (brown planthopper)); Laode/phax spp.(e.g., L. striatellus (small brown planthopper)); Nephotettix spp.(e.g., N. virescens or N. cincticeps (green leafhopper), or N.nigropictus (rice leafhopper)); Sogatella spp. (e.g., S. furcifera(white-backed planthopper)); Chilo spp. (e.g., C. suppressalis (ricestriped stem borer), C. auricilius (gold-fringed stem borer), or C.polychrysus (dark-headed stem borer)); Sesamia spp. (e.g., S. inferens(pink rice borer)); Tryporyza spp. (e.g., T. innotata (white riceborer), or T. incertulas (yellow rice borer)); Anthonomus spp. (e.g., A.grandis (boll weevil)); Phaedon spp. (e.g., P. cochleariae (mustard leafbeetle)); Epilachna spp. (e.g., E. varivetis (Mexican bean beetle));Tribolium spp. (e.g., T. castaneum (red floor beetle)); Diabrotica spp.(e.g., D. virgifera (western corn rootworm), D. barberi (northern cornrootworm), D. undecimpunctata howardi (southern corn rootworm), D.virgifera zeae (Mexican corn rootworm); Ostrinia spp. (e.g., O.nubilalis (European corn borer)); Anaphothrips spp. (e.g., A. obscrurus(grass thrips)); Pectinophora spp. (e.g., P. gossypiella (pinkbollworm)); Heliothis spp. (e.g., H. virescens (tobacco budworm));Trialeurodes spp. (e.g., T. abutiloneus (banded-winged whitefly) T.vaporariorum (greenhouse whitefly)); Bemisia spp. (e.g., B. argentifolii(silverleaf whitefly)); Aphis spp. (e.g., A. gossypii (cotton aphid));Lygus spp. (e.g., L. lineolaris (tarnished plant bug) or L. hesperus(western tarnished plant bug)); Euschistus spp. (e.g., E. conspersus(consperse stink bug)); Chlorochroa spp. (e.g., C. sayi (Say stinkbug));Nezara spp. (e.g., N. viridula (southern green stinkbug)); Thrips spp.(e.g., T. tabaci (onion thrips)); Frankliniella spp. (e.g., F. fusca(tobacco thrips), or F. occidentalis (western flower thrips)); Achetaspp. (e.g., A. domesticus (house cricket)); Myzus spp. (e.g., M.persicae (green peach aphid)); Macrosiphum spp. (e.g., M. euphorbiae(potato aphid)); Blissus spp. (e.g., B. leucopterus (chinch bug));Acrosternum spp. (e.g., A. hilare (green stink bug)); Chilotraea spp.(e.g., C. polychrysa (rice stalk borer)); Lissorhoptrus spp. (e.g., L.oryzophilus (rice water weevil)); Rhopalosiphum spp. (e.g., R. maidis(corn leaf aphid)); and Anuraphis spp. (e.g., A. maidiradicis (corn rootaphid)).

The endophyte-associated plant can be tested for its ability to resist,or otherwise repel, pathogenic insects by measuring, for example,overall plant biomass, biomass of the fruit or grain, percentage ofintact leaves, or other physiological parameters described herein, andcomparing with a reference agricultural plant. In one embodiment, theendophyte-associated plant exhibits at least 5% greater biomass, forexample, at least 10%, at least 15%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% or more biomass,than the reference agricultural plant grown under the same conditions(e.g., grown side-by-side, or adjacent to, the endophyte-associatedplants). In other embodiments, the endophyte-associated plant exhibitsat least 5% greater fruit or grain yield, for example, at least 10%, atleast 15%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 75%, at least 100% or more fruit or grain yield, than thereference agricultural plant grown under the same conditions (e.g.,grown side-by-side, or adjacent to, the endophyte-associated plants).

Nematodes.

Nematodes are microscopic roundworms that feed on the roots, fluids,leaves and stems of more than 2,000 row crops, vegetables, fruits, andornamental plants, causing an estimated $100 billion crop loss worldwideand accounting for 13% of global crop losses due to disease. A varietyof parasitic nematode species infect crop plants, including root-knotnematodes (RKN), cyst- and lesion-forming nematodes. Root-knotnematodes, which are characterized by causing root gall formation atfeeding sites, have a relatively broad host range and are thereforeparasitic on a large number of crop species. The cyst- andlesion-forming nematode species have a more limited host range, butstill cause considerable losses in susceptible crops.

Signs of nematode damage include stunting and yellowing of leaves, andwilting of the plants during hot periods. Nematode infestation, however,can cause significant yield losses without any obvious above-grounddisease symptoms. The primary causes of yield reduction are due tounderground root damage. Roots infected by SCN are dwarfed or stunted.Nematode infestation also can decrease the number of nitrogen-fixingnodules on the roots, and may make the roots more susceptible to attacksby other soil-borne plant nematodes.

In one embodiment, the endophyte-associated plant has an increasedresistance to a nematode when compared with a reference agriculturalplant. As before with insect herbivores, biomass of the plant or aportion of the plant, or any of the other physiological parametersmentioned elsewhere, can be compared with the reference agriculturalplant grown under the same conditions. Particularly useful measurementsinclude overall plant biomass, biomass and/or size of the fruit orgrain, and root biomass. In one embodiment, the endophyte-associatedplant exhibits at least 5% greater biomass, for example, at least 10%,at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 75%, at least 100% or more biomass, than the referenceagricultural plant grown under the same conditions (e.g., grownside-by-side, or adjacent to, the endophyte-associated plants, underconditions of nematode challenge). In another embodiment, theendophyte-associated plant exhibits at least 5% greater root biomass,for example, at least 10%, at least 15%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% or more rootbiomass, than the reference agricultural plant grown under the sameconditions (e.g., grown side-by-side, or adjacent to, theendophyte-associated plants, under conditions of nematode challenge). Instill another embodiment, the endophyte-associated plant exhibits atleast 5% greater fruit or grain yield, for example, at least 10%, atleast 15%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 75%, at least 100% or more fruit or grain yield, than thereference agricultural plant grown under the same conditions (e.g.,grown side-by-side, or adjacent to, the endophyte-associated plants,under conditions of nematode challenge).

Fungal Pathogens.

Fungal diseases are responsible for yearly losses of over $10 Billion onagricultural crops in the US, represent 42% of global crop losses due todisease, and are caused by a large variety of biologically diversepathogens. Different strategies have traditionally been used to controlthem. Resistance traits have been bred into agriculturally importantvarieties, thus providing various levels of resistance against either anarrow range of pathogen isolates or races, or against a broader range.However, this involves the long and labor intensive process ofintroducing desirable traits into commercial lines by genetic crossesand, due to the risk of pests evolving to overcome natural plantresistance, a constant effort to breed new resistance traits intocommercial lines is required. Alternatively, fungal diseases have beencontrolled by the application of chemical fungicides. This strategyusually results in efficient control, but is also associated with thepossible development of resistant pathogens and can be associated with anegative impact on the environment. Moreover, in certain crops, such asbarley and wheat, the control of fungal pathogens by chemical fungicidesis difficult or impractical.

The present invention contemplates the use of endophytes which are ableto confer resistance to fungal pathogens to the host plant. Increasedresistance to fungal inoculation can be measured, for example, using anyof the physiological parameters presented above, by comparing withreference agricultural plants. In one embodiment, theendophyte-associated plant exhibits at least 5% greater biomass, forexample, at least 10%, at least 15%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% or more biomass,than the reference agricultural plant grown under the same conditions(e.g., grown side-by-side, or adjacent to, the endophyte-associatedplants, infected with the fungal pathogen). In still another embodiment,the endophyte-associated plant exhibits at least 5% greater fruit orgrain yield, for example, at least 10%, at least 15%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 75%, at least 100% ormore fruit or grain yield, than the reference agricultural plant grownunder the same conditions (e.g., grown side-by-side, or adjacent to, theendophyte-associated plants, infected with the fungal pathogen). Inanother embodiment, the endophyte-associated plant exhibits at least a5% reduction in for hyphal growth, for example, at least 10%, at least15%, at least 20%, at least 30%, at least 40%, at least 50%, at least75%, at least 90% reduction or more, in hyphal growth, than thereference agricultural plant grown under the same conditions (e.g.,grown side-by-side, or adjacent to, the endophyte-associated plants,infected with the fungal pathogen).

Viral Pathogens.

Plant viruses are estimated to account for 18% of global crop losses dueto disease. There are numerous examples of viral pathogens affectingagricultural productivity. Examples include the American wheat striatemosaic virus (AWSMV) (wheat striate mosaic), Barley stripe mosaic virus(BSMV), Barley yellow dwarf virus (BYDV), Brome mosaic virus (BMV),Cereal chlorotic mottle virus (CCMV), Corn chlorotic vein banding virus(CCVBV), Brazilian maize mosaic virus, Corn lethal necrosis Viruscomplex from Maize chlorotic mottle virus, (MCMV), Maize dwarf mosaicvirus (MDMV), A or B Wheat streak mosaic virus (WSMV), Cucumber mosaicvirus (CMV), Cynodon chlorotic streak virus (CCSV), Johnsongrass mosaicvirus (JGMV), Maize bushy stunt Mycoplasma-like organism (MLO)associated virus, Maize chlorotic dwarf Maize chlorotic dwarf virus(MCDV), Maize chlorotic mottle virus (MCMV), Maize dwarf mosaic virus(MDMV), strains A, D, E and F, Maize leaf fleck virus (MLFV), Maize linevirus (MLV), Maize mosaic (corn leaf stripe, Maize mosaic virus (MMV),enanismo rayado), Maize mottle and chlorotic stunt virus, Maize pellucidringspot virus (MPRV), Maize raya gruesa virus (MRGV), Maize rayado fino(fine striping) virus (MRFV), Maize red stripe virus (MRSV), Maize ringmottle virus (MRMV), Maize rio cuarto virus (MRCV), Maize rough dwarfvirus (MRDV), Cereal tillering disease virus, Maize sterile stunt virus,barley yellow striate virus, Maize streak virus (MSV), Maize stripevirus, Maize chloroticstripe virus, maize hoja blanca virus, Maizestunting virus; Maize tassel abortion virus (MTAV), Maize vein enationvirus (MVEV), Maize wallaby ear virus (MWEV), Maize white leaf virus,Maize white line mosaic virus (MWLMV), Millet red leaf virus (MRLV),Northern cereal mosaic virus (NCMV), Oat pseudorosette virus,(zakuklivanie), Oat sterile dwarf virus (OSDV), Rice black-streakeddwarf virus (RBSDV), Rice stripe virus (RSV), Sorghum mosaic virus(SrMV), Sugarcane mosaic virus (SCMV) strains H, 1 and M, Sugarcane Fijidisease virus (FDV), Sugarcane mosaic virus (SCMV) strains A, B, D, E,SC, BC, Sabi and MB (formerly MDMV-B), and Wheat spot mosaic virus(WSMV). In one embodiment, the endophyte-associated plant providesprotection against viral pathogens such that there is at least 5%greater biomass, for example, at least 10%, at least 15%, at least 20%,at least 30%, at least 40%, at least 50%, at least 75%, at least 100% ormore biomass, than the reference agricultural plant grown under the sameconditions. In still another embodiment, the endophyte-associated plantexhibits at least 5% greater fruit or grain yield, for example, at least10%, at least 15%, at least 20%, at least 30%, at least 40%, at least50%, at least 75%, at least 100% or more fruit or grain yield whenchallenged with a virus, than the reference agricultural plant grownunder the same conditions. In yet another embodiment, theendophyte-associated plant exhibits at least 5% lower viral titer, forexample, at least 10%, at least 15%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% lower viral titerwhen challenged with a virus, than the reference agricultural plantgrown under the same conditions.

Bacterial Pathogens.

Likewise, bacterial pathogens are a significant problem negativelyaffecting agricultural productivity and accounting for 27% of globalcrop losses due to plant disease. In one embodiment, theendophyte-associated plant described herein provides protection againstbacterial pathogens such that there is at least 5% greater biomass, forexample, at least 10%, at least 15%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% or more biomass,than the reference agricultural plant grown under the same conditions.In still another embodiment, the endophyte-associated plant exhibits atleast 5% greater fruit or grain yield, for example, at least 10%, atleast 15%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 75%, at least 100% or more fruit or grain yield when challengedwith a bacterial pathogen, than the reference agricultural plant grownunder the same conditions. In yet another embodiment, theendophyte-associated plant exhibits at least 5% lower bacterial count,for example, at least 10%, at least 15%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% lower bacterialcount when challenged with a bacteria, than the reference agriculturalplant grown under the same conditions.

Improvement of Other Traits.

In other embodiments, the inoculated endophyte can confer otherbeneficial traits to the plant. Improved traits can include an improvednutritional content of the plant or plant part used for humanconsumption. In one embodiment, the endophyte-associated plant is ableto produce a detectable change in the content of at least one nutrient.Examples of such nutrients include amino acid, protein, oil (includingany one of Oleic acid, Linoleic acid, Alpha-linolenic acid, Saturatedfatty acids, Palmitic acid, Stearic acid and Trans fats), carbohydrate(including sugars such as sucrose, glucose and fructose, starch, ordietary fiber), Vitamin A, Thiamine (vit. B1), Riboflavin (vit. B2),Niacin (vit. B3), Pantothenic acid (B5), Vitamin B6, Folate (vit. B9),Choline, Vitamin C, Vitamin E, Vitamin K, Calcium, Iron, Magnesium,Manganese, Phosphorus, Potassium, Sodium, Zinc. In one embodiment, theendophyte-associated plant or part thereof contains at least 10% morenutrient, for example, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100%, at least 150%, at least 200%, at least 300% or more, of thenutrient when compared with reference agricultural plants.

In other cases, the improved trait can include reduced content of aharmful or undesirable substance when compared with referenceagricultural plants. Such compounds include those which are harmful wheningested in large quantities or are bitter tasting (for example, oxalicacid, amygdalin, certain alkaloids such as solanine, caffeine, nicotine,quinine and morphine, tannins, cyanide). As such, in one embodiment, theendophyte-associated plant or part thereof contains at least 10% less ofthe undesirable substance, for example, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, at least 99% less of theundesirable substance when compared with reference agricultural plant.

In a related embodiment, the improved trait can include improved tasteof the plant or a part of the plant, including the fruit or seed. In arelated embodiment, the improved trait can include reduction ofundesirable compounds produced by other endophytes in plants, such asdegradation of Fusarium produced deoxynivalenol (also known as vomitoxinand a virulence factor involved in Fusarium head blight of maize andwheat) in a part of the plant, including the fruit or seed.

In other cases, the improved trait can be an increase in overall biomassof the plant or a part of the plant, including its fruit or seed.

The endophyte-associated plant can also have an altered hormone statusor altered levels of hormone production when compared with a referenceagricultural plant. An alteration in hormonal status may affect manyphysiological parameters, including flowering time, water efficiency,apical dominance and/or lateral shoot branching, increase in root hair,and alteration in fruit ripening.

The association between the endophyte and the plant can also be detectedusing other methods known in the art. For example, the biochemical,metabolomics, proteomic, genomic, epigenomic and/or trasncriptomicprofiles of endophyte-associated plants can be compared with referenceagricultural plants under the same conditions.

Metabolomic differences between the plants can be detected using methodsknown in the art. For example, a biological sample (whole tissue,exudate, phloem sap, xylem sap, root exudate, etc.) from theendophyte-associated and reference agricultural plants can be analyzedessentially as described in Fiehn et al., (2000) Nature Biotechnol., 18,1157-1161, or Roessner et al., (2001) Plant Cell, 13, 11-29. Suchmetabolomic methods can be used to detect differences in levels inhormone, nutrients, secondary metabolites, root exudates, phloem sapcontent, xylem sap content, heavy metal content, and the like. Suchmethods are also useful for detecting alterations in microbial contentand status; for example, the presence and levels of bacterial/fungalsignaling molecules (e.g., autoinducers and pheromones), which canindicate the status of group-based behavior of endophytes based on, forexample, population density (see, for example Daniels et al., (2006).PNAS 103: 14965-14970. Eberhard et al., (1981). Biochemistry 20 (9):2444-2449). Transcriptome analysis (reviewed, for example, in Usadel &Fernie, (2013). Front Plant Sci. 4:48) of endophyte-associated andreference agricultural plants can also be performed to detect changes inexpression of at least one transcript, or a set or network of genes uponendophyte association. Similarly, epigenetic changes can be detectedusing methylated DNA immunoprecipitation followed by high-throughputsequencing (Vining et al., (2013) BMC Plant Biol. 13:92).

Combinations of Endophytic Microbes

Combinations of endophytic microbes such as seed-origin bacterialendophytes can be selected by any one or more of several criteria. Inone embodiment, compatible endophytic populations are selected. As usedherein, compatibility refers to microbial populations which do notsignificantly interfere with the growth and propagation of the other.Incompatible microbial populations can arise, for example, where one ofthe populations produces or secrets a compound which is toxic ordeleterious to the growth of the other population(s). Incompatibilityarising from production of deleterious compounds/agents can be detectedusing methods known in the art, and as described herein elsewhere.Similarly, the distinct populations can compete for limited resources ina way that makes co-existence difficult.

In another embodiment, combinations are selected on the basis ofcompounds produced by each population. For example, the first populationis capable of producing siderophores, and another population is capableof producing anti-fungal compounds. In one embodiment, the firstpopulation of bacterial endophytes is capable of a function selectedfrom the group consisting of auxin production, nitrogen fixation,production of an antimicrobial compound, siderophore production, mineralphosphate solubilization, cellulase production, chitinase production,xylanase production, and acetoin production. In another embodiment, thesecond population of bacterial endophytes is capable of a functionselected from the group consisting of auxin production, nitrogenfixation, production of an antimicrobial compound, siderophoreproduction, mineral phosphate solubilization, cellulase production,chitinase production, xylanase production, and acetoin production. Instill another embodiment, the first and second populations are capableof at least one different function.

In still another embodiment, the combinations are selected which displaydistinct localization in the plant after colonization. For example, thefirst population can colonize, and in some cases preferentiallycolonize, the root tissue, while a second population can be selected onthe basis of its preferential colonization of the aerial parts of theagricultural plant. Therefore, in one embodiment, the first populationis capable of colonizing one or more of the tissues selected from thegroup consisting of a root, shoot, leaf, flower, and seed. In anotherembodiment, the second population is capable of colonizing one or moretissues selected from the group consisting of root, shoot, leaf, flower,and seed. In still another embodiment, the first and second populationsare capable of colonizing a different tissue within the agriculturalgrass plant.

In still another embodiment, the combinations of endophytes are selectedwhich confer one or more distinct fitness traits on the inoculatedagricultural plant, either individually or in synergistic associationwith other endophytes. Alternatively, two or more endophytes induce thecolonization of a third endophyte. For example, the first population isselected on the basis that it confers significant increase in biomass,while the second population promotes increased drought tolerance on theinoculated agricultural plant. Therefore, in one embodiment, the firstpopulation is capable of conferring at least one trait selected from thegroup consisting of thermal tolerance, herbicide tolerance, droughtresistance, insect resistance, fungus resistance, virus resistance,bacteria resistance, male sterility, cold tolerance, salt tolerance,increased yield, enhanced nutrient use efficiency, increased nitrogenuse efficiency, increased fermentable carbohydrate content, reducedlignin content, increased antioxidant content, enhanced water useefficiency, increased vigor, increased germination efficiency, earlieror increased flowering, increased biomass, altered root-to-shoot biomassratio, enhanced soil water retention, or a combination thereof. Inanother embodiment, the second population is capable of conferring atrait selected from the group consisting of thermal tolerance, herbicidetolerance, drought resistance, insect resistance, fungus resistance,virus resistance, bacteria resistance, male sterility, cold tolerance,salt tolerance, increased yield, enhanced nutrient use efficiency,increased nitrogen use efficiency, increased fermentable carbohydratecontent, reduced lignin content, increased antioxidant content, enhancedwater use efficiency, increased vigor, increased germination efficiency,earlier or increased flowering, increased biomass, altered root-to-shootbiomass ratio, enhanced soil water retention, or a combination thereof.In still another embodiment, each of the first and second population iscapable of conferring a different trait selected from the groupconsisting of thermal tolerance, herbicide tolerance, droughtresistance, insect resistance, fungus resistance, virus resistance,bacteria resistance, male sterility, cold tolerance, salt tolerance,increased yield, enhanced nutrient use efficiency, increased nitrogenuse efficiency, increased fermentable carbohydrate content, reducedlignin content, increased antioxidant content, enhanced water useefficiency, increased vigor, increased germination efficiency, earlieror increased flowering, increased biomass, altered root-to-shoot biomassratio, enhanced soil water retention, or a combination thereof.

The combinations of endophytes can also be selected based oncombinations of the above criteria. For example, the first populationcan be selected on the basis of the compound it produces (e.g., itsability to fix nitrogen, thus providing a potential nitrogen source tothe plant), while the second population is selected on the basis of itsability to confer increased resistance of the plant to a pathogen (e.g.,a fungal pathogen).

Formulations/Seed Coating Compositions

The purified bacterial populations described herein can be formulatedusing an agriculturally compatible carrier. The formulation useful forthese embodiments generally typically include at least one memberselected from the group consisting of a tackifier, a microbialstabilizer, a fungicide, an antibacterial agent, an herbicide, anematicide, an insecticide, a plant growth regulator, a rodenticide, adessicant, and a nutrient.

In some cases, the purified bacterial population is mixed with anagriculturally compatible carrier. The carrier can be a solid carrier orliquid carrier, and in various forms including microspheres, powders,emulsions and the like. The carrier may be any one or more of a numberof carriers that confer a variety of properties, such as increasedstability, wettability, or dispersability. Wetting agents such asnatural or synthetic surfactants, which can be nonionic or ionicsurfactants, or a combination thereof can be included in a compositionof the invention. Water-in-oil emulsions can also be used to formulate acomposition that includes the purified bacterial population (see, forexample, U.S. Pat. No. 7,485,451, which is incorporated herein byreference in its entirety). Suitable formulations that may be preparedinclude wettable powders, granules, gels, agar strips or pellets,thickeners, and the like, microencapsulated particles, and the like,liquids such as aqueous flowables, aqueous suspensions, water-in-oilemulsions, etc. The formulation may include grain or legume products,for example, ground grain or beans, broth or flour derived from grain orbeans, starch, sugar, or oil.

In some embodiments, the agricultural carrier may be soil or a plantgrowth medium. Other agricultural carriers that may be used includewater, fertilizers, plant-based oils, humectants, or combinationsthereof. Alternatively, the agricultural carrier may be a solid, such asdiatomaceous earth, loam, silica, alginate, clay, bentonite,vermiculite, seed cases, other plant and animal products, orcombinations, including granules, pellets, or suspensions. Mixtures ofany of the aforementioned ingredients are also contemplated as carriers,such as but not limited to, pesta (flour and kaolin clay), agar orflour-based pellets in loam, sand, or clay, etc. Formulations mayinclude food sources for the cultured organisms, such as barley, rice,or other biological materials such as seed, plant parts, sugar canebagasse, hulls or stalks from grain processing, ground plant material orwood from building site refuse, sawdust or small fibers from recyclingof paper, fabric, or wood. Other suitable formulations will be known tothose skilled in the art.

In one embodiment, the formulation can include a tackifier or adherent.Such agents are useful for combining the bacterial population of theinvention with carriers that can contain other compounds (e.g., controlagents that are not biologic), to yield a coating composition. Suchcompositions help create coatings around the plant or seed to maintaincontact between the microbe and other agents with the plant or plantpart. In one embodiment, adherents are selected from the groupconsisting of: alginate, gums, starches, lecithins, formononetin,polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinylacetate, cephalins, Gum Arabic, Xanthan Gum, Mineral Oil, PolyethyleneGlycol (PEG), Polyvinyl pyrrolidone (PVP), Arabino-galactan, MethylCellulose, PEG 400, Chitosan, Polyacrylamide, Polyacrylate,Polyacrylonitrile, Glycerol, Triethylene glycol, Vinyl Acetate, GellanGum, Polystyrene, Polyvinyl, Carboxymethyl cellulose, Gum Ghatti, andpolyoxyethylene-polyoxybutylene block copolymers. Other examples ofadherent compositions that can be used in the synthetic preparationinclude those described in EP 0818135, CA 1229497, WO 2013090628, EP0192342, WO 2008103422 and CA 1041788, each of which is incorporatedherein by reference in its entirety.

The formulation can also contain a surfactant. Non-limiting examples ofsurfactants include nitrogen-surfactant blends such as Prefer 28(Cenex), Surf-N(US), Inhance (Brandt), P-28 (Wilfarm) and Patrol(Helena); esterified seed oils include Sun-It II (AmCy), MSO (UAP),Scoil (Agsco), Hasten (Wilfarm) and Mes-100 (Drexel); andorgano-silicone surfactants include Silwet L77 (UAP), Silikin (Terra),Dyne-Amic (Helena), Kinetic (Helena), Sylgard 309 (Wilbur-Ellis) andCentury (Precision). In one embodiment, the surfactant is present at aconcentration of between 0.01% v/v to 10% v/v. In another embodiment,the surfactant is present at a concentration of between 0.1% v/v to 1%v/v.

In certain cases, the formulation includes a microbial stabilizer. Suchan agent can include a desiccant. As used herein, a “desiccant” caninclude any compound or mixture of compounds that can be classified as adesiccant regardless of whether the compound or compounds are used insuch concentrations that they in fact have a desiccating effect on theliquid inoculant. Such desiccants are ideally compatible with thebacterial population used, and should promote the ability of themicrobial population to survive application on the seeds and to survivedesiccation. Examples of suitable desiccants include one or more oftrehalose, sucrose, glycerol, and Methylene glycol. Other suitabledesiccants include, but are not limited to, non reducing sugars andsugar alcohols (e.g., mannitol or sorbitol). The amount of desiccantintroduced into the formulation can range from about 5% to about 50% byweight/volume, for example, between about 10% to about 40%, betweenabout 15% and about 35%, or between about 20% and about 30%.

In some cases, it is advantageous for the formulation to contain agentssuch as a fungicide, an antibacterial agent, an herbicide, a nematicide,an insecticide, a plant growth regulator, a rodenticide, or a nutrient.Such agents are ideally compatible with the agricultural seed orseedling onto which the formulation is applied (e.g., it should not bedeleterious to the growth or health of the plant). Furthermore, theagent is ideally one which does not cause safety concerns for human,animal or industrial use (e.g., no safety issues, or the compound issufficiently labile that the commodity plant product derived from theplant contains negligible amounts of the compound).

In the liquid form, for example, solutions or suspensions, the bacterialendophytic populations of the present invention can be mixed orsuspended in water or in aqueous solutions. Suitable liquid diluents orcarriers include water, aqueous solutions, petroleum distillates, orother liquid carriers.

Solid compositions can be prepared by dispersing the bacterialendophytic populations of the invention in and on an appropriatelydivided solid carrier, such as peat, wheat, bran, vermiculite, clay,talc, bentonite, diatomaceous earth, fuller's earth, pasteurized soil,and the like. When such formulations are used as wettable powders,biologically compatible dispersing agents such as non-ionic, anionic,amphoteric, or cationic dispersing and emulsifying agents can be used.

The solid carriers used upon formulation include, for example, mineralcarriers such as kaolin clay, pyrophyllite, bentonite, montmorillonite,diatomaceous earth, acid white soil, vermiculite, and pearlite, andinorganic salts such as ammonium sulfate, ammonium phosphate, ammoniumnitrate, urea, ammonium chloride, and calcium carbonate. Also, organicfine powders such as wheat flour, wheat bran, and rice bran may be used.The liquid carriers include vegetable oils such as soybean oil andcottonseed oil, glycerol, ethylene glycol, polyethylene glycol,propylene glycol, polypropylene glycol, etc.

In one particular embodiment, the formulation is ideally suited forcoating of the endophytic microbial population onto seeds. The bacterialendophytic populations described in the present invention are capable ofconferring many fitness benefits to the host plants. The ability toconfer such benefits by coating the bacterial populations on the surfaceof seeds has many potential advantages, particularly when used in acommercial (agricultural) scale.

The bacterial endophytic populations herein can be combined with one ormore of the agents described above to yield a formulation suitable forcombining with an agricultural seed or seedling. The bacterialpopulation can be obtained from growth in culture, for example, using asynthetic growth medium. In addition, the microbe can be cultured onsolid media, for example on petri dishes, scraped off and suspended intothe preparation. Microbes at different growth phases can be used. Forexample, microbes at lag phase, early-log phase, mid-log phase, late-logphase, stationary phase, early death phase, or death phase can be used.

The formulations comprising the bacterial endophytic population of thepresent invention typically contains between about 0.1 to 95% by weight,for example, between about 1% and 90%, between about 3% and 75%, betweenabout 5% and 60%, between about 10% and 50% in wet weight of thebacterial population of the present invention. It is preferred that theformulation contains at least about 10³ CFU per ml of formulation, forexample, at least about 10⁴, at least about 10⁵, at least about 10⁶, atleast 10⁷ CFU, at least 10⁸ CFU per ml of formulation.

Population of Seeds

In another aspect, the invention provides for a substantially uniformpopulation of seeds comprising a plurality of seeds comprising thebacterial endophytic population, as described herein above. Substantialuniformity can be determined in many ways. In some cases, at least 10%,for example, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 75%, at least 80%, at least 90%, atleast 95% or more of the seeds in the population, contains the bacterialendophytic population in an amount effective to colonize the plantdisposed on the surface of the seeds. In other cases, at least 10%, forexample, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 75%, at least 80%, at least 90%, atleast 95% or more of the seeds in the population, contains at least 1,10, or 100 CFU on the seed surface or per gram of seed, for example, atleast 200 CFU, at least 300 CFU, at least 1,000 CFU, at least 3,000 CFU,at least 10,000 CFU, at least 30,000 CFU, at least 100,000 CFU, at least300,000 CFU, or at least 1,000,000 CFU per seed or more.

In a particular embodiment, the population of seeds is packaged in a bagor container suitable for commercial sale. Such a bag contains a unitweight or count of the seeds comprising the bacterial endophyticpopulation as described herein, and further comprises a label. In oneembodiment, the bag or container contains at least 1,000 seeds, forexample, at least 5,000 seeds, at least 10,000 seeds, at least 20,000seeds, at least 30,000 seeds, at least 50,000 seeds, at least 70,000seeds, at least 80,000 seeds, at least 90,000 seeds or more. In anotherembodiment, the bag or container can comprise a discrete weight ofseeds, for example, at least 1 lb, at least 2 lbs, at least 5 lbs, atleast 10 lbs, at least 30 lbs, at least 50 lbs, at least 70 lbs or more.The bag or container comprises a label describing the seeds and/or saidbacterial endophytic population. The label can contain additionalinformation, for example, the information selected from the groupconsisting of: net weight, lot number, geographic origin of the seeds,test date, germination rate, inert matter content, and the amount ofnoxious weeds, if any. Suitable containers or packages include thosetraditionally used in plant seed commercialization. The invention alsocontemplates other containers with more sophisticated storagecapabilities (e.g., with microbiologically tight wrappings or withgas-or water-proof containments).

In some cases, a sub-population of seeds comprising the bacterialendophytic population is further selected on the basis of increaseduniformity, for example, on the basis of uniformity of microbialpopulation. For example, individual seeds of pools collected fromindividual cobs, individual plants, individual plots (representingplants inoculated on the same day) or individual fields can be testedfor uniformity of microbial density, and only those pools meetingspecifications (e.g., at least 80% of tested seeds have minimum density,as determined by quantitative methods described elsewhere) are combinedto provide the agricultural seed sub-population.

The methods described herein can also comprise a validating step. Thevalidating step can entail, for example, growing some seeds collectedfrom the inoculated plants into mature agricultural plants, and testingthose individual plants for uniformity. Such validating step can beperformed on individual seeds collected from cobs, individual plants,individual plots (representing plants inoculated on the same day) orindividual fields, and tested as described above to identify poolsmeeting the required specifications.

In some embodiments, methods described herein include planting asynthetic combination described herein. Suitable planters include an airseeder and/or fertilizer apparatus used in agricultural operations toapply particulate materials including one or more of the following,seed, fertilizer and/or inoculants, into soil during the plantingoperation. Seeder/fertilizer devices can include a tool bar havingground-engaging openers thereon, behind which is towed a wheeled cartthat includes one or more containment tanks or bins and associatedmetering means to respectively contain and meter therefrom particulatematerials. See, e.g., U.S. Pat. No. 7,555,990.

In certain embodiments, a composition described herein may be in theform of a liquid, a slurry, a solid, or a powder (wettable powder or drypowder). In another embodiment, a composition may be in the form of aseed coating. Compositions in liquid, slurry, or powder (e.g., wettablepowder) form may be suitable for coating seeds. When used to coat seeds,the composition may be applied to the seeds and allowed to dry. Inembodiments wherein the composition is a powder (e.g., a wettablepowder), a liquid, such as water, may need to be added to the powderbefore application to a seed.

In still another embodiment, the methods can include introducing intothe soil an inoculum of one or more of the endophyte populationsdescribed herein. Such methods can include introducing into the soil oneor more of the compositions described herein. The inoculum(s) orcompositions may be introduced into the soil according to methods knownto those skilled in the art. Non-limiting examples include in-furrowintroduction, spraying, coating seeds, foliar introduction, etc. In aparticular embodiment, the introducing step comprises in-furrowintroduction of the inoculum or compositions described herein.

In one embodiment, seeds may be treated with composition(s) describedherein in several ways but preferably via spraying or dripping. Sprayand drip treatment may be conducted by formulating compositionsdescribed herein and spraying or dripping the composition(s) onto aseed(s) via a continuous treating system (which is calibrated to applytreatment at a predefined rate in proportion to the continuous flow ofseed), such as a drum-type of treater. Batch systems, in which apredetermined batch size of seed and composition(s) as described hereinare delivered into a mixer, may also be employed. Systems and apparatifor performing these processes are commercially available from numeroussuppliers, e.g., Bayer CropScience (Gustafson).

In another embodiment, the treatment entails coating seeds. One suchprocess involves coating the inside wall of a round container with thecomposition(s) described herein, adding seeds, then rotating thecontainer to cause the seeds to contact the wall and the composition(s),a process known in the art as “container coating”. Seeds can be coatedby combinations of coating methods. Soaking typically entails usingliquid forms of the compositions described. For example, seeds can besoaked for about 1 minute to about 24 hours (e.g., for at least 1 min, 5min, 10 min, 20 min, 40 min, 80 min, 3 hr, 6 hr, 12 hr, 24 hr).

Population of Plants/Agricultural Fields

A major focus of crop improvement efforts has been to select varietieswith traits that give, in addition to the highest return, the greatesthomogeneity and uniformity. While inbreeding can yield plants withsubstantial genetic identity, heterogeneity with respect to plantheight, flowering time, and time to seed, remain impediments toobtaining a homogeneous field of plants. The inevitable plant-to-plantvariability are caused by a multitude of factors, including unevenenvironmental conditions and management practices. Another possiblesource of variability can, in some cases, be due to the heterogeneity ofthe microbial population inhabit the plants. By providing bacterialendophytic populations onto seeds and seedlings, the resulting plantsgenerated by germinating the seeds and seedlings have a more consistentmicrobial composition, and thus are expected to yield a more uniformpopulation of plants.

Therefore, in another aspect, the invention provides a substantiallyuniform population of plants. The population can include at least 100plants, for example, at least 300 plants, at least 1,000 plants, atleast 3,000 plants, at least 10,000 plants, at least 30,000 plants, atleast 100,000 plants or more. The plants are grown from the seedscomprising the bacterial endophytic population as described herein. Theincreased uniformity of the plants can be measured in a number ofdifferent ways.

In one embodiment, there is an increased uniformity with respect to themicrobes within the plant population. For example, in one embodiment, asubstantial portion of the population of plants, for example at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 90%, at least95% or more of the seeds or plants in a population, contains a thresholdnumber of the bacterial endophytic population. The threshold number canbe at least 10 CFU, at least 100 CFU, for example at least 300 CFU, atleast 1,000 CFU, at least 3,000 CFU, at least 10,000 CFU, at least30,000 CFU, at least 100,000 CFU or more, in the plant or a part of theplant. Alternatively, in a substantial portion of the population ofplants, for example, in at least 1%, at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 90%, at least 95% or more of theplants in the population, the bacterial endophyte population that isprovided to the seed or seedling represents at least 0.1%, at least 1%,at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, or 100% of the total microbe population in theplant/seed.

In one embodiment, there is increased genetic uniformity of asubstantial proportion or all detectable microbes within the taxa,genus, or species of the seed-origin microbe relative to an uninoculatedcontrol. This increased uniformity can be a result of the seed-originmicrobe being of monoclonal origin or otherwise deriving from aseed-origin microbial population comprising a more uniform genomesequence and plasmid repertoire than would be present in the microbialpopulation a plant that derives its microbial community largely viaassimilation of diverse soil symbionts.

In another embodiment, there is an increased uniformity with respect toa physiological parameter of the plants within the population. In somecases, there can be an increased uniformity in the height of the plantswhen compared with a population of reference agricultural plants grownunder the same conditions. For example, there can be a reduction in thestandard deviation in the height of the plants in the population of atleast 5%, for example, at least 10%, at least 15%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60% or more, whencompared with a population of reference agricultural plants grown underthe same conditions. In other cases, there can be a reduction in thestandard deviation in the flowering time of the plants in the populationof at least 5%, for example, at least 10%, at least 15%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60% or more, whencompared with a population of reference agricultural plants grown underthe same conditions.

Commodity Plant Product

The present invention provides a commodity plant product, as well asmethods for producing a commodity plant product, that is derived from aplant of the present invention. As used herein, a “commodity plantproduct” refers to any composition or product that is comprised ofmaterial derived from a plant, seed, plant cell, or plant part of thepresent invention. Commodity plant products may be sold to consumers andcan be viable or nonviable. Nonviable commodity products include but arenot limited to nonviable seeds and grains; processed seeds, seed parts,and plant parts; dehydrated plant tissue, frozen plant tissue, andprocessed plant tissue; seeds and plant parts processed for animal feedfor terrestrial and/or aquatic animal consumption, oil, meal, flour,flakes, bran, fiber, paper, tea, coffee, silage, crushed of whole grain,and any other food for human or animal consumption; and biomasses andfuel products; and raw material in industry. Industrial uses of oilsderived from the agricultural plants described herein includeingredients for paints, plastics, fibers, detergents, cosmetics,lubricants, and biodiesel fuel. Soybean oil may be split,inter-esterified, sulfurized, epoxidized, polymerized, ethoxylated, orcleaved. Designing and producing soybean oil derivatives with improvedfunctionality and improved oliochemistry is a rapidly growing field. Thetypical mixture of triglycerides is usually split and separated intopure fatty acids, which are then combined with petroleum-derivedalcohols or acids, nitrogen, sulfonates, chlorine, or with fattyalcohols derived from fats and oils to produce the desired type of oilor fat. Commodity plant products also include industrial compounds, suchas a wide variety of resins used in the formulation of adhesives, films,plastics, paints, coatings and foams.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Isolation of Bacterial Endophytes from Seeds,Seedlings, and Plants

In order to better understand the role played by seed-borne endophyticmicrobes in improving the vigor, general health and stress resilience ofhost plants, a systematic screen was initiated to isolate andcharacterize endophytic microbes from seeds of commercially significantgrass plants.

Diverse types of maize, wheat, rice, and other seeds were acquired andscreened for cultivatable microbes. 49 distinct cultivars of maize andteosinte accessions were sourced from the USDA via GRIN (NationalGenetic Resources Program at http://www.ars-grin.gov/) or purchased fromthe Sustainable Seed Company (Covelo, Calif.). Similarly, 5 distinctwheat cultivars and wheat relatives were sourced from the USDA via GRIN(National Genetic Resources Program at http://www.ars-grin.gov/) orpurchased from a Whole Foods in Cambridge, Mass. Seeds of rice and ricerelatives (23 in total) were sourced from the USDA via GRIN (NationalGenetic Resources Program at http://www.ars-grin.gov/) or purchased froma Whole Foods in Cambridge, Mass. Seeds of several other species ofplants, including sorghum, millet, oat, rye, teff, etc., were sourcedfrom the USDA via GRIN (National Genetic Resources Program at the worldwide web at ars-grin.gov/), the Sustainable Seed Company or purchasedfrom a Whole Foods in Cambridge, Mass.

Pools of 5 seeds were soaked in 10 mL of sterile water contained insterile 15 mL conical tubes for 24 hours. Some maize and rice accessionswere sampled for seed surface microbes. In these cases, after 24 hoursof soaking, 50 μL aliquots of undiluted, 100× dilute and 10000× dilutesoaking water was plated onto R2A agar [Proteose peptone (0.5 g/L),Casamino acids (0.5 g/L), Yeast extract (0.5 g/L), Dextrose (0.5 g/L)Soluble starch (0.5 g/L), Dipotassium phosphate (0.3 g/L), Magnesiumsulfate 7H₂O (0.05 g/L), Sodium pyruvate (0.3 g/L), Agar (15 g/L), FinalpH 7±0.2 @ 25° C.] to culture oligotrophic bacteria, while the samevolumes and dilutions were also plated onto potato dextrose agar (PDA)[Potato Infusion from 200 g/L, Dextrose 20 g/L, Agar 15 g/L, Final pH:5.6±0.2 at 25° C.] to culture copiotrophic bacteria and fungi. All seedsin the study were sampled for endophytes by surface sterilization,trituration, and culturing of the mash. Seeds were surface sterilized bywashing with 70% EtOH, rinsing with water, then washing with a 3%solution of sodium hypochlorite followed by 3 rinses in sterile water.All wash and rinse steps were 5 minutes with constant shaking at 130rpm. Seeds were then blotted on R2A agar which was incubated at 30° C.for 7 days in order to confirm successful surface sterilization.Following the sterilization process, batches of seeds were ground with asterile mortar and pestle in sterile R2A broth, while seeds of maize,rice and soy were also grown in sterile conditions and the roots orshoots of seedlings (without further sterilization) were crushed by beadbeating in a Fastprep24 machine with 3 carbide beads, 1 mL of R2A brothin a 15 mL Falcon tube shaking at 6M/s for 60 seconds. Extracts ofsurface washes, crushed seed, or macerated seedling tissue were seriallydiluted by factors of 1 to 10⁻³ and spread onto quadrants on R2A, PDA,LGI or V8 juice agar in order to isolate cultivable seed-bornemicroorganisms. Plates were incubated at 28° C. for 7 days, monitoringfor the appearance of colonies daily. After a week, plates werephotographed and different morphotypes of colonies were identified andlabeled. These were then selected for identification by sequencing,backing up by freezing at −80° C. as glycerol stock, and assaying forbeneficial functions as described herein.

Plating and Scoring of Microbes

After 7 days of growth, most bacterial colonies had grown large anddistinct enough to allow differentiation based on colony size, shape,color and texture. Photographs of each plate were taken, and on thebasis of color and morphotype, different colonies were identified bynumber for later reference. These strains were also streaked out ontoeither R2A or PDA to check for purity, and clean cultures were thenscraped with a loop off the plate, resuspended in a mixture of R2A andglycerol, and frozen away in quadruplicate at −80° C. for later.

Example 2 Sequence Analysis & Phylogenetic Assignment

To accurately characterize the isolated bacterial endophytes, colonieswere submitted for marker gene sequencing, and the sequences wereanalyzed to provide taxonomic classifications. Colonies were subjectedto 16S rRNA gene PCR amplification using a 27f/1492r primer set, andSanger sequencing of paired ends was performed at Genewiz (SouthPlainfield, N.J.). Raw chromatograms were converted to sequences, andcorresponding quality scores were assigned using TraceTuner v3.0.6beta(U.S. Pat. No. 6,681,186, incorporated herein by reference). Thesesequences were quality filtered using PRINSEQ v0.20.3 [Schmieder andEdwards (2011) Bioinformatics. 2011; 27:863-864, incorporated herein byreference] with left and right trim quality score thresholds of 30 and aquality window of 20 bp. Sequences without paired reads were discardedfrom further processing. Paired end quality filtered sequences weremerged using USEARCH v7.0 [Edgar (2010) Nature methods 10:996-8].Taxonomic classifications were assigned to the sequences using the RDPclassifier [Wang et al., (2007) Applied and environmental microbiology73:5261-7, incorporated herein by reference] trained on the Greengenesdatabase [McDonald et al. (2012), ISME journal 6:610-8, incorporatedherein by reference]. The resulting 473 microbes, representing 44distinct OTUs (using a 97% similarity threshold) are provided in Table2.

Example 3 In-Vitro Testing of Bacterial Endophytes

A total of 242 seed-origin bacterial endophytes representing 44 distinctOTUs as described above were seeded onto 96 well plates and tested forvarious activities and/or production of compounds, as described below.Colonies or wells with no detectable activity were scored as “-”, lowactivity as “1,” moderate activity as “2” and strong activity as “3.”The results of these in vitro assays are summarized in Table 3.

Production of Auxin (SD)

To allow isolates to grow and accumulate auxin, bacterial strains wereinoculated into 250 μL of R2A broth supplemented with with L-tryptophan(5 mM) in 350 μL deep, transparent flat bottom, 96 well culture plates.The plates were sealed with a breathable membrane and incubated at 28°C. under static conditions for 3 days. After 3 days the OD600 and OD530nm were measured on a plate reader to check for bacterial growth. Aftermeasuring these ODs, 50 μL of yellowish Salkowski reagent (0.01 M FeCl3in 35% HClO4 (perchloric acid, #311421, Sigma) were added to each welland incubated in the dark for 30 minutes before measuring the OD530 nmmeasured to detect pink/red color.

Auxin is an important plant hormone, which can promote cell enlargementand inhibit branch development (meristem activity) in above ground planttissues, while below ground it has the opposite effect, promoting rootbranching and growth. Interestingly, plant auxin is manufactured aboveground and transported to the roots. It thus follows that plant andespecially root inhabiting microbes which produce significant amounts ofauxin will be able to promote root branching and development even underconditions where the plant reduces its own production of auxin. Suchconditions can exist for example when soil is flooded and rootsencounter an anoxic environment which slows or stops root metabolism.

Seed-origin bacteria were screened for their ability to produce auxinsas possible root growth promoting agents. A very large number ofbacteria showed a detectable level of pink or red colour development(the diagnostic feature of the assay suggesting auxin or indoliccompound production)—169 out of 247. 89 strains had particularly strongproduction of auxin or indole compounds. Erwinia and Pantoea species arevery similar if not identical taxonomic groups and can thus beconsidered together—of a total of 38 isolates, 23 had moderate or strongproduction of auxin or indole compounds in vitro. Many of these Erwiniaand Pantoea strains were isolated from inside surface sterilized seeds,suggesting they may be able to colonize the inside of the emerging root(first plant part to emerge the seed) and stimulate root growth for byproducing auxins on the inside of the plant.

Another important group of auxin producing seed-origin bacteria werePseudomonas species, 9 of the 14 isolated showed significant productionof indoles in this assay. Ochrobactrum species were also detected asstrong producers of indolic compounds in this assay, with 15 of 18showing moderate to strong color change (although all 18 had detectablecolour change in this assay). Strongly auxin producing strains ofPseudomonas and Ochrobactrum species were isolated from seed surfaces.

Mineral Phosphate Solubilization

Microbes were plated on tricalcium phosphate media as described inRodriguez et al., (2001) J Biotechnol 84: 155-161 (incorporated hereinby reference). This was prepared as follows: 10 g/L glucose, 0.373 g/LNH₄NO₃, 0.41 g/L MgSO₄, 0.295 g/L NaCl, 0.003 FeCl₃, 0.7 g/L Ca₃HPO₄,100 mM Tris and 20 g/L Agar, pH 7, then autoclaved and poured intosquare Petri plates. After 3 days of growth at 28° C. in darkness, clearhalos were measured around colonies able to solubilize the tricalciumphosphate. This was an agar based assay looking for halos aroundcolonies which signify the solubilization of opaque tri-calciumphosphate, which resulted in a large number (95) of isolates havingdetectable levels of phosphate solubilization (Table 3). Of these, atleast 36 had moderate to high levels of phosphate solubilization,including several Enterobacter and Pantoea species.

Growth on Nitrogen Free LGI Media

All glassware was cleaned with 6 M HCl before media preparation. A new96 well plate (300 ul well volume) was filled with 250 ul/well ofsterile LGI broth [per L, 50 g Sucrose, 0.01 g FeCl₃-6H₂O, 0.8 g K₃PO₄,0.2 g CaCl2, 0.2 g MgSO₄-7H₂O, 0.002 g Na₂MoO₄-2H₂O, pH 7.5]. Bacteriawere inoculated into the 96 wells simultaneously with a flame-sterilized96 pin replicator. The plate was sealed with a breathable membrane,incubated at 28° C. without shaking for 5 days, and OD₆₀₀ readings takenwith a 96 well plate reader.

A nitrogen fixing plant associated bacterium is able theoretically toadd to the host's nitrogen metabolism, and the most famous beneficialplant associated bacteria, rhizobia, are able to do this withinspecially adapted organs on the roots of leguminous plants. The seedassociated bacteria in this study may be able to fix nitrogen inassociation with the developing seedling, whether they colonize theplant's surfaces or interior and thereby add to the plant's nitrogennutrition.

In total, of the 247 isolates there were 34 (14%) which had detectablegrowth under nitrogen limiting conditions (Table 3).

TABLE 3 Functional assays to examine the potential for seed-originmicrobes to confer novel functions to crops. Shows Shows Growth on ACCSEQ ID Antagonizes Antagonizes Cellulolytic Pectinolytic SecretesPhosphate Nitrogen Deaminase Produces Produces Sym Strain ID OTU # NO:Taxonomy E. coli S. cerevisciae activity activity siderophoresSolubilization Free LGI Activity Auxin/Indoles Acetoin SYM00033 0 541Enterobacter sp. — — 1 1 1 2 — — 3 — SYM00173 0 593 Pantoea sp. 2 — 1 1— 2 Yes — 3 1 SYM00176 0 596 Pantoea sp. 1 — 1 1 2 1 — — 2 1 SYM00605 0716 — — 1 1 2 2 — — 1 — SYM00607 0 717 — — — — 2 2 — — 1 2 SYM00608 0718 Pantoea sp. — — — — 1 — — 1 1 1 SYM00620 0 720 Enterobacter sp. — 11 1 — 1 — — 2 2 SYM00658 0 736 1 1 1 1 — 2 — 1 2 3 SYM00660 1 737Pseudomonas sp. — 1 2 2 1 — — 1 — 1 SYM00011 2 522 Pseudomonas sp. — — —— — 1 Yes — 2 — SYM00011b 2 523 Pseudomonas sp. — — — — — — — — — 1SYM00013 2 524 Pseudomonas sp. — — 2 2 2 — Yes — 2 — SYM00014 2 526Pseudomonas sp. — — 2 2 1 — Yes — 2 — SYM00062 2 557 Pseudomonas sp. — —2 2 2 — — 1 2 — SYM00068 2 563 Pseudomonas sp. — — 2 2 2 1 — 3 2 —SYM00069 2 564 Pseudomonas sp. — — — — — — — — — 2 SYM00646 2 730Pseudomonas sp. — — 2 2 3 — — — 2 — SYM00649 2 733 Pseudomonas sp. — — 22 1 — — 3 2 — SYM00650 2 734 Pseudomonas sp. — 1 2 2 — — — 3 2 —SYM00657 2 735 Pseudomonas sp. — — 2 2 — — — 3 2 — SYM00672 2 738Pseudomonas sp. — — 2 2 2 1 — 3 1 — SYM00709 2 747 Pseudomonas sp. — — 33 — — — — — 3 SYM00013b 3 525 Curtobacterium sp. — — — — — — — — — 1SYM00167 3 588 Curtobacterium sp. — — — — — — — — 1 — SYM00171 3 591Curtobacterium sp. — — — — 2 — — — 1 — SYM00174 3 594 Curtobacterium sp.— — — — — — — — 1 1 SYM00178 3 598 Curtobacterium sp. — — 1 1 1 — — — —1 SYM00180 3 600 Curtobacterium sp. — — — — — — — — — 1 SYM00181 3 601Curtobacterium sp. — — — — — — — — — 2 SYM00235 3 622 Curtobacterium sp.— — 1 1 — 1 Yes — 3 3 SYM00244 3 626 Curtobacterium sp. — — 1 1 — 1 — —— 1 SYM00525 3 654 Curtobacterium sp. — — — — — — — — 2 1 SYM00625 3 724Curtobacterium sp. — — 2 2 — — — 1 1 — SYM00645 3 729 Curtobacterium sp.— — — — 3 — — 3 1 — SYM00647 3 731 Curtobacterium sp. — — 1 1 — — — — 13 SYM00690 3 740 Curtobacterium sp. — — — — — — — 1 1 1 SYM00691 3 741Curtobacterium sp. — — — — — — — 1 — 1 SYM00693 3 742 Curtobacterium sp.— — 1 1 — — — 1 — 1 SYM00712 3 748 Curtobacterium sp. — — 1 1 — — — 1 1— SYM00716 3 752 Curtobacterium sp. — — — — — — — 1 1 1 SYM00722 3 753Curtobacterium sp. — — 1 1 — — — 1 1 — SYM00731B 3 756 Curtobacteriumsp. — — — — — — — 1 1 — SYM00784 3 773 Curtobacterium sp. 2 — — — — — —— 1 — SYM00188 6 605 Paenibacillus sp. — — — — — — — — — 2 SYM00190 6607 Paenibacillus sp. — — 1 1 — 1 — — — — SYM00195 6 610 Paenibacillussp. — — — — — 2 — — — 2 SYM00217 6 616 Paenibacillus sp. — — — — — 2 — —— — SYM00227 6 619 Paenibacillus sp. — — 1 1 — 1 — 1 — — SYM00597 6 711Paenibacillus sp. — — — — — 1 — — — 3 SYM00017b 7 532 Pantoea sp. — — 11 — 2 — — 3 — SYM00018 7 534 Pantoea sp. — — — — — — — — 2 — SYM00020 7535 Pantoea sp. — — — — — 1 Yes — 3 — SYM00022 7 537 Pantoea sp. — — 1 11 — — — 2 — SYM00025 7 538 Pantoea sp. — — 1 1 — — — — 2 1 SYM00043 7544 Pantoea sp. — — 1 1 1 2 Yes — 1 — SYM00047 7 546 Pantoea sp. — — 1 1— 2 — — 1 1 SYM00049 7 547 Pantoea sp. — — — — 1 — — — 3 1 SYM00055 7553 Pantoea sp. — — 1 1 1 2 — — — — SYM00057 7 554 Pantoea sp. — — — — —— — — — 1 SYM00058 7 555 Pantoea sp. — — — — — — — — — 3 SYM00078 7 568Pantoea sp. 3 1 1 1 1 2 Yes — 3 — SYM00081 7 569 Pantoea sp. — — 1 1 1 2Yes — 1 — SYM00082a 7 570 Pantoea sp. — — — — 1 — Yes — 1 — SYM00085 7571 Pantoea sp. — — 1 1 1 2 — — 1 1 SYM00086 7 572 Pantoea sp. — — 1 1 12 — — 1 1 SYM00088 7 574 Pantoea sp. — — — — — — — — — 3 SYM00094 7 576Pantoea sp. — — 1 1 1 2 Yes — 1 1 SYM00095 7 577 Pantoea sp. — — 1 1 1 2Yes — 1 1 SYM00096 7 578 Pantoea sp. — — 1 1 1 — — — 1 1 SYM00100 7 579Pantoea sp. 1 1 1 1 1 1 — — 3 — SYM00101 7 580 Pantoea sp. — — — — 1 — —— 2 — SYM00502 7 639 Erwinia sp. — — — — 1 1 — — 3 — SYM00506 7 641Erwinia sp. — — 1 1 1 1 — — 3 1 SYM00506b 7 642 Erwinia sp. — 1 1 1 1 1— — 3 3 SYM00511 7 647 Erwinia sp. — — — — — — — — 2 1 SYM00514b 7 649Erwinia sp. — — — — — 2 — — 3 3 SYM00514C 7 650 Erwinia sp. — — — — — —— 3 — 1 SYM00514D 7 651 Erwinia sp. — — — — — — — — 2 3 SYM00731A 7 755Erwinia sp. — — 1 1 — 1 — 1 2 — SYM00785 7 774 Erwinia sp. 1 1 1 1 — 2 —1 2 — SYM00544 9 663 Ochrobactrum sp. — 1 — — — 1 — — 3 — SYM00545B 9665 Ochrobactrum sp. — 1 — — — — — — 2 — SYM00548 9 667 Ochrobactrum sp.— 1 — — — 1 — — 2 — SYM00552 9 670 Ochrobactrum sp. — 1 — — — — — — 2 1SYM00558 9 675 Ochrobactrum sp. — 1 — — — 1 — — 2 — SYM00580b 9 689Ochrobactrum sp. — 1 — — — — — — 1 — SYM00580d 9 691 Ochrobactrum sp. —1 — — — — — — 2 — SYM00583 9 699 Ochrobactrum sp. — 1 — — — 1 — — 2 —SYM00584 9 700 Ochrobactrum sp. — — — — — 1 — — 2 — SYM00588 9 705Ochrobactrum sp. — 1 — — — 2 — — 2 2 SYM00596 9 710 Ochrobactrum sp. — 1— — — 1 — — 2 3 SYM00600 9 713 Ochrobactrum sp. — 1 — — — 2 — — 2 —SYM00746 9 757 Ochrobactrum sp. 1 1 — — — 1 — 1 1 1 SYM00752 9 759Ochrobactrum sp. 1 1 — — — 1 — 1 2 — SYM00756 9 761 Ochrobactrum sp. 1 —— — — 1 — 1 1 — SYM00763 9 767 Ochrobactrum sp. 1 — — — — 1 — — 2 —SYM00783 9 772 Ochrobactrum sp. 1 1 — — — 1 — — 2 — SYM00812 9 775Ochrobactrum sp. — — — — — — — — 2 — SYM00064a 10 560 Stenotrophomonas —— — — — — — — 1 — sp. SYM00183 10 603 Stenotrophomonas — — — — — — — — 12 sp. SYM00184 10 604 Stenotrophomonas — — — — — — — — 1 3 sp. SYM0054312 662 Bacillus sp. 1 1 — — — — — — 1 — SYM00595 12 709 Bacillus sp. 1 1— — — — — — 1 — SYM00580C 13 690 Achromobacter sp. — — — — 1 — — 1 1 —SYM00547 13 666 Achromobacter sp. — — — — 2 — — 1 1 — SYM00551 13 669Achromobacter sp. — 1 — — 1 — — 2 1 — SYM00560 13 676 Achromobacter sp.— — — — 1 — — — 2 — SYM00565B 13 681 Achromobacter sp. — — — — 1 1 — 1 11 SYM00580i 13 694 Achromobacter sp. — 1 — — — — — — 1 — SYM00585 13 701Achromobacter sp. — — — — 1 2 — 1 2 — SYM00586b 13 702 Achromobacter sp.— 1 — — 2 — — — 2 — SYM00588b 13 706 Achromobacter sp. — — — — — — — — 32 SYM00591 13 708 Achromobacter sp. — — — — — — — 3 1 — SYM00602 13 715Achromobacter sp. — — — — 3 — — — 1 2 SYM00758 13 763 Achromobacter sp.— — — — — — — 3 1 — SYM00761 13 765 Achromobacter sp. — — — — 1 — — 1 —— SYM00764 13 768 Achromobacter sp. — — — — 1 — — 1 1 — SYM00765 13 769Achromobacter sp. — — — — — — — — — 3 SYM00824 13 777 Achromobacter sp.— 1 — — — — — 3 1 — SYM00828 13 778 Achromobacter sp. — — — — 1 — — — 1— SYM00830 13 779 Achromobacter sp. — — — — — — — 3 1 — SYM00831 13 780Achromobacter sp. — — — — 1 1 — 1 1 — SYM00028 18 540 Enterobacter sp. 11 1 1 — 1 — — 1 3 SYM00052 18 550 Enterobacter sp. — — 1 1 — 1 — — 1 1SYM00053 18 551 Enterobacter sp. — — 1 1 — 1 — — — 1 SYM00054 18 552Enterobacter sp. — — — — 1 — — — — 3 SYM00175 18 595 Enterobacter sp. —— 1 1 1 2 Yes — 1 — SYM00627 18 725 Enterobacter sp. 1 2 1 1 — 2 — 1 — 3SYM00715 18 751 Enterobacter sp. — — — — — 2 — 1 — 2 SYM00189 19 606Bacillus sp. — — — — — — — — — 1 SYM00192 19 608 Bacillus sp. — — — — —— — — — — SYM00197 19 611 Bacillus sp. — — — — — — — — 1 2 SYM00201 19612 Bacillus sp. — — — — — — — — 1 — SYM00202 19 613 Bacillus sp. — — —— — 2 — — — — SYM00215 19 615 Bacillus sp. — — — — — — — — — 3 SYM0023319 621 Bacillus sp. — — — — — — Yes — 2 1 SYM00016b 25 529Methylobacterium — — 1 1 — — — — 1 1 sp. SYM00236 25 623Methylobacterium — — 1 1 — 1 Yes 1 — — sp. SYM00237 25 624Methylobacterium — — 1 1 — 1 Yes 1 2 — sp. SYM00240 25 625Methylobacterium — — 1 1 — 1 Yes 3 — — sp. SYM00501 27 638 Burkholderiasp. 3 1 — — 2 — — 3 2 — SYM00504 27 640 Burkholderia sp. 3 1 — — 2 — — 32 — SYM00536 27 656 Burkholderia sp. 3 1 — — 3 1 — 1 2 — SYM00538E 27659 Burkholderia sp. 1 1 — — 2 1 — 3 1 — SYM00566A 27 682 Burkholderiasp. 2 1 — — 2 — — 3 — 3 SYM00568 27 683 Burkholderia sp. 2 1 — — 2 — — 31 — SYM00570 27 684 Burkholderia sp. 2 1 — — 2 1 — 3 1 — SYM00574 27 685Burkholderia sp. 2 1 — — 2 1 — 3 1 1 SYM00575 27 686 Burkholderia sp. 31 — — 2 1 — 3 1 — SYM00578 27 687 Burkholderia sp. 2 1 — — 2 2 — 3 — —SYM00621 27 721 Burkholderia sp. 1 1 — — 3 — — 3 1 — SYM00623 27 722Burkholderia sp. 1 1 — — 3 — — 3 — — SYM00624 27 723 Burkholderia sp. 11 — — 3 — — 3 — — SYM00633 27 727 Burkholderia sp. 1 1 1 1 — 2 — 1 3 3SYM00822 27 776 Burkholderia sp. — — — — 3 1 — — — — SYM00037 28 543Bacillus sp. — — — — — — — — — 2 SYM00051 28 549 Microbacterium sp. — 2— — 2 — — — 2 2 SYM00104 28 582 Microbacterium sp. 1 — — — — — Yes — — —SYM00177 28 597 Microbacterium sp. — — — — — — — — 1 3 SYM00514A 28 648Microbacterium sp. — — — — — — — — 2 2 SYM00523 28 652 Microbacteriumsp. — — — — — — — — 2 2 SYM00538H 28 660 Microbacterium sp. — — — — — —— — — 2 SYM00542 28 661 Microbacterium sp. — — 1 1 — — — — 1 1 SYM0055628 674 Microbacterium sp. — — 1 1 — — — — 3 — SYM00581A 28 695Microbacterium sp. — — — — — — — — 2 3 SYM00586c 28 703 Microbacteriumsp. — — 1 1 — — — — 2 2 SYM00587 28 704 Microbacterium sp. — — 2 2 — — —— 2 1 SYM00598 28 712 Microbacterium sp. — — — — — — — — 1 2 SYM00757 28762 Microbacterium sp. — — — — — — — 1 — 3 SYM00760 28 764Microbacterium sp. — — — — — — — 1 — 2 SYM00780 28 771 Microbacteriumsp. — — — — 1 — — — 1 — SYM00832 28 781 Microbacterium sp. 1 — — — — — —— — 1 SYM00015 29 528 Xanthomonas sp. 1 — 2 2 2 — Yes — 1 1 SYM00021 29536 Xanthomonas sp. 2 — 3 3 2 — — — 2 — SYM00179 29 599 Xanthomonas sp.1 — 2 2 — 1 — — 1 1 SYM00182 29 602 Xanthomonas sp. 1 — 1 1 — 1 — 1 3 3SYM00252 29 630 Xanthomonas sp. — — — — — — Yes — — — SYM00565A 30 680Rhodococcus sp. — 1 — — — 1 — — — — SYM00580G 30 693 Rhodococcus sp. — 1— — 2 1 — — 1 — SYM00753 30 760 Rhodococcus sp. 1 1 — — — — Yes 1 1 2SYM00762 30 766 Rhodococcus sp. 1 — — — 1 1 Yes — 1 — SYM00775 30 770Rhodococcus sp. 1 1 — — 2 1 Yes 1 1 — SYM00589 31 707 Paenibacillus sp.— — — — — — — — 3 2 SYM00057B 37 1446 Burkholderia — 1 1 1 1 1 Yes 3 1 —phytofirmans SYM00102 38 581 Staphylococcus sp. — — — — — — — — — 2SYM00072 39 566 Bacillus sp. 2 — — — — — — — — 3 SYM00075 39 567Bacillus sp. 2 — — — — — — — — 3 SYM00249 39 628 Bacillus sp. — — — — —— — — — — SYM00507 39 645 Bacillus sp. 2 1 — — — — — — 2 1 SYM00553 39671 Bacillus sp. — 1 — — — — — — — 1 SYM00562 39 677 Bacillus sp. 2 — —— — — — — — — SYM00564 39 679 Bacillus sp. 2 1 — — — — — — — — SYM00580E39 692 Bacillus sp. — 1 — — 1 — — — — 1 SYM00581b 39 696 Bacillus sp. 2— — — — — — — 2 3 SYM00581c 39 697 Bacillus sp. — — — — — — — 1 1 3SYM00601 39 714 Bacillus sp. 1 — — — — — — — — 3 SYM00036 41 542Bacillus sp. 3 2 — — — — — — — 3 SYM00110 41 586 Bacillus sp. 3 1 — — —— Yes — 1 — SYM00193 41 609 Bacillus sp. 3 — — — — — — — — 1 SYM00218 41617 Bacillus sp. 3 1 — — — 1 — — — — SYM00250 41 629 Bacillus sp. — 1 —— — 1 Yes — — — SYM00697 41 745 Bacillus sp. 3 3 — — — — — — — 3SYM00704 41 746 Bacillus sp. 3 3 — — — — — — — 3 SYM00017c 45 533Sphingomonas sp. — — 1 1 — — Yes — 2 1 SYM00062b 45 558 Sphingomonas sp.— — 1 1 — — — — 3 1 SYM00065 45 561 Sphingomonas sp. — — — — — — — — — 1SYM00168 45 589 Sphingomonas sp. — 1 2 2 — 2 Yes — 2 1 SYM00169 45 590Sphingomonas sp. — 1 2 2 — 2 Yes — 3 3 SYM00231 46 620 Sphingobium sp. —1 2 2 1 2 Yes — 2 — SYM00975 51 843 Herbaspirillum sp. — — — — 2 2 — — —3 SYM00506c 53 643 Paenibacillus sp. — — — — — — — — 3 1 SYM00506D 53644 Paenibacillus sp. — — — — — — — — 2 — SYM00545 53 664 Paenibacillussp. — 1 — — — — — — 2 — SYM00549 53 668 Paenibacillus sp. — — — — — — —— 1 — SYM00554 53 672 Paenibacillus sp. — 1 — — — — — — 1 1 SYM00555 53673 Paenibacillus sp. — 1 — — — — — — — — SYM00012 55 1447Microbacterium 1 — — — — 1 — — 1 1 binotii SYM00046 56 545 Enterobactersp. 1 3 1 1 2 1 — — 1 3 SYM00050 56 548 Enterobacter sp. — 2 1 1 1 1 — —2 2 SYM00628 56 726 Enterobacter sp. 1 1 1 1 — 1 — 1 3 3 SYM00106 59 583Micrococcus sp. — — 1 1 — — Yes — — — SYM00107 59 584 Micrococcus sp. —— — — — — Yes — — 1 SYM00108 59 585 Micrococcus sp. — — 1 1 — — Yes — —— SYM00090 62 575 Chryseobacterium sp. 1 — — — 1 — — — — — SYM00002 66521 Agrobacterium sp. — — 2 2 — — — — 3 — SYM00017a 66 531 Agrobacteriumsp. — — 2 2 — — — — 3 — SYM00714 66 750 Agrobacterium sp. — — 1 1 — — —1 2 — SYM00060 67 556 Staphylococcus sp. — — — — — — — — — 3 SYM00071 76565 Bacillus sp. — — — — — — — — — 2 SYM00204 76 614 Bacillus sp. — — —— — — — — — — SYM00563 76 678 Bacillus sp. — — — — — — — — — — SYM0061776 719 Bacillus sp. — — — — — — — — 1 2 SYM00960 82 831 Luteibacter sp.— — — — 2 — — — — 3 SYM00940 83 815 — — — — — 1 — — — 3 SYM00713 84 749Erwinia sp. — 1 1 1 1 1 — 1 2 1 SYM00992 126 856 Sphingomonas sp. — — —— — 2 — — — 2 SYM00063 134 559 Microbacterium sp. 1 — — — — — — — 1 3SYM00226 134 618 Microbacterium sp. — — — — — — — — — — SYM00246 134 627Microbacterium sp. — 1 — — — — — — 1 1 SYM00524 134 653 Microbacteriumsp. — — — — — — — — 1 3 SYM00199 135 1448 Bacillus sp. — — — — — 2 — — —— SYM00172 146 592 Pantoea sp. 2 — 1 1 3 2 Yes — 3 3 SYM00527 146 655Erwinia sp. — — 1 1 — 1 — — 3 1 SYM00644 146 728 Erwinia sp. — — 1 1 1 1— 3 2 2 SYM00648 146 732 1 1 — — 1 2 — 1 1 3 SYM00538A 172 658Sphingomonas sp. — — 1 1 — — — — 2 — SYM00508 196 646 — — 1 1 — 1 — — 2— Legend: “—” indicates no significant increase; “1” = low activity; “2”= medium activity; “3” = high activity

All of these groups are known to have representatives with the potentialto fix atmospheric nitrogen; however chief among these were Bacillus,Burkholderia, Enterobacter, Methylobacteria, and Pseudomonas.

Seed-origin isolates growing on N Free Genus Media Bacillus sp. 3Burkholderia sp. 1 Curtobacterium sp. 1 Enterobacter sp. 1Methylobacterium sp. 3 Microbacterium sp. 1 Micrococcus sp. 3 Pantoeasp. 9 Pseudomonas sp. 3 Rhodococcus sp. 3 Sphingobium sp. 1 Sphingomonassp. 3 Xanthomonas sp. 2

ACC Deaminase Activity

Microbes were assayed for growth with ACC as their sole source ofnitrogen.

Prior to media preparation all glassware was cleaned with 6 M HCl. A 2 Mfilter sterilized solution of ACC (#1373A, Research Organics, USA) wasprepared in water. 2 μl/mL of this was added to autoclaved LGI broth(see above), and 250 μL aliquots were placed in a brand new (clean) 96well plate. The plate was inoculated with a 96 pin library replicator,sealed with a breathable membrane, incubated at 28° C. without shakingfor 5 days, and OD600 readings taken. Only wells that were significantlymore turbid than their corresponding nitrogen free LGI wells wereconsidered to display ACC deaminase activity.

Plant stress reactions are strongly impacted by the plant's ownproduction and overproduction of the gaseous hormone ethylene. Ethyleneis metabolized from its precursor 1-aminocyclopropane-1-carboxylate(ACC) which can be diverted from ethylene metabolism by microbial andplant enzymes having ACC deaminase activity. As the name implies, ACCdeaminase removes molecular nitrogen from the ethylene precursor,removing it as a substrate for production of the plant stress hormoneand providing for the microbe a source of valuable nitrogen nutrition.It is somewhat surprising, but this microbial ability to inhibitethylene production is very important for plant health as damage togrowth and productivity under various stress conditions is believed toresult from the plant's own over-production of ethylene (Journal ofIndustrial Microbiology & Biotechnology, October 2007, Volume 34, Issue10, pp 635-648).

In total, of the 247 isolates there were 68 (28%) which had greatergrowth on nitrogen free LGI media supplemented with ACC, than innitrogen free LGI. Of these, only 11% had very high ACC deaminaseactivity and these were mostly strains of Achromobacter, Burkholderia,and Pseudomonas. Chief amongst these were Burkholderia species whichheld ACC deaminase as their most distinctive in vitro characteristic—94%or 15 out of 16 Burkholderia isolates had ACC deaminase activity. OfBurkholderia isolates, 81% had strong ACC deaminase activity, while only42% of Achromobacter species (5 of 12 isolates) had strong ACC deaminaseactivity, and next were Pseudomonas where only 5 of 14 isolates (42%)had strong activity. Many Curtobacteria isolates appeared to have ACCdeaminase activity as well, however these were all rated low (as 1) andthus of less interest than the preceeding groups of isolates.

Seed-Origin Isolates growing on ACC Genus as the sole Nitrogen SourceAchromobacter sp. 12 Agrobacterium sp. 1 Bacillus sp. 1 Burkholderia sp.15 Curtobacterium sp. 9 Enterobacter sp. 3 Erwinia sp. 5Methylobacterium sp. 3 Microbacterium sp. 2 Ochrobactrum sp. 3 Pantoeasp. 1 Pseudomonas sp. 7 Rhodococcus sp. 2 Xanthomonas sp. 1

Acetoin and Diacetyl Production

The method was adapted from Phalip et al., (1994) J Basic Microbiol 34:277-280. (incorporated herein by reference). 250 ml of autoclaved R2Abroth supplemented with 0.5% glucose was aliquoted into a 96 well plate(#07-200-700, Fisher). The bacterial endophytes from a glycerol stockplate were inoculated into the plate using a flame-sterilized 96 pinreplicator, sealed with a breathable membrane, then incubated for 3 dayswithout shaking at 28° C. At day 5, 50 μl/well was added of freshlyblended Barritt's Reagents A and B [5 g/L creatine mixed 3:1 (v/v) withfreshly prepared ∝-naphthol (75 g/L in 2.5 M sodium hydroxide)]. After15 minutes, plates were scored for red or pink coloration relative to acopper colored negative control (measured as 525 nm absorption on aplate reader).

A large number of seed-origin bacteria showed a detectable level of pinkor red color development (126 out of 247; See Table 3). 70 of 247isolates had strong production of acetoin or butanediol as detected bythis assay. Bacillus (13 of 33), Enterobacter (8 or 16) andMicrobacterium (12 of 21) species were the most intense producers ofacetoin/butanediol in this collection. In addition, two of the threeisolates of Stenotrophomonas included in this study were also strongacetoin/butanediol producers.

Siderophore Production

To ensure no contaminating iron was carried over from previousexperiments, all glassware was deferrated with 6 M HCl and water priorto media preparation [Cox (1994) Methods Enzymol 235: 315-329,incorporated herein by reference]. In this cleaned glassware, R2A brothmedia, which is iron limited, was prepared and poured (250 ul/well) into96 well plates and the plate then inoculated with bacteria using a 96pin plate replicator. After 3 days of incubation at 28° C. withoutshaking, to each well was added 100 ul of O-CAS preparation withoutgelling agent [Perez-Miranda et al. (2007), J Microbiol Methods 70:127-131, incorporated herein by reference]. One liter of O-CAS reagentwas prepared using the cleaned glassware by mixing 60.5 mg of chromeazurol S (CAS), 72.9 mg of hexadecyltrimethyl ammonium bromide (HDTMA),30.24 g of finely crushed Piperazine-1,4-bis-2-ethanesulfonic acid(PIPES) with 10 ml of 1 mM FeCl₃.6H₂O in 10 mM HCl solvent. The PIPEShad to be finely powdered and mixed gently with stirring (not shaking)to avoid producing bubbles, until a deep blue color was achieved. 15minutes after adding the reagent to each well, color change was scoredby looking for purple halos (catechol type siderophores) or orangecolonies (hydroxamate siderophores) relative to the deep blue of theO-CAS.

Siderophore production by bacteria on a plant surface or inside a plantmay both show that a microbe is equipped to grow in a nutrient limitedenvironment, and perhaps protect the plant environment from invasion byother, perhaps undesirable microbes. We searched for two types ofsiderophore which result in purple color change (catechol typesiderophores) or orange color change (hydroxamate siderophores) afteraddition of the blue O-Cas reagent to 96 well plates. A large number ofbacteria showed a detectable level of color change relative to the deepblue of the O-CAS; 80 out of 247. Notably, 32 of 247 strains had strongproduction of siderophores. Interestingly, strong siderophore producersincluded a large number (14) of the 16 Burkholderia isolates. Manyisolates of Achromobacter (9 of 12) and Pantoea (15 of 26) were able toinduce weak colour change in the O-CAS material.

Seed-origin Isolates Producing Strong Genus Siderophores Achromobactersp. 3 Burkholderia sp. 14 Curtobacterium sp. 2 Enterobacter sp. 1Microbacterium sp. 1 Pantoea sp. 2 Pseudomonas sp. 5 Rhodococcus sp. 2Xanthomonas sp. 2

Pectinase Activity

Iodine reacts with pectin to form a dark blue-colored complex, leavingclear halos as evidence of extracellular enzyme activity. Adapting aprevious protocol [Soares et al. (1999) Rev de Microbiol 30: 299-303,incorporated herein by reference] 0.2% (w/v) of citrus pectin (#76280,Sigma) and 0.1% triton X-100 were added to R2A media, autoclaved andpoured into 150 mm plates. Bacteria were inoculated using a 96 pin platereplicator. After 3 days of culturing in the darkness at 25° C.,pectinase activity was visualized by flooding the plate with Gram'siodine. Positive colonies were surrounded by clear halos. In our study,a large number, roughly 83 of the 247 isolates, had detectable pectinaseactivity, and 21 of these isolates had moderate to strong resultsvisualized as medium to large halos—caused by copious diffusion ofenzyme away from the bacteria.

Cellulase Activity

Iodine reacts with cellulose to form a dark brown/blue-colored complex,leaving clear halos as evidence of extracellular enzyme activity.Adapting a previous protocol [Kasana et al. (2008), Curr Microbiol 57:503-507, incorporated herein by reference] 0.2% carboxymethylcellulose(CMC) sodium salt (#C5678, Sigma) and 0.1% triton X-100 were added to astarch free variant of R2A media, autoclaved and poured into 150 mmplates. Bacteria were inoculated using a 96 pin plate replicator. After3 days of culturing in the darkness at 25° C., cellulose activity wasvisualized by flooding the plate with Gram's iodine. Positive colonieswere surrounded by clear halos.

In our study, a large number, roughly 83 of the 247 isolates, haddetectable cellulose activity, and 21 of these isolates had moderate tostrong results visualized as medium to large halos—caused by copiousdiffusion of enzyme away from the bacteria.

Antibiosis

Briefly, colonies of either E. coli DH5α (bacterial tester) or yeaststrain Saccharomyces cerevisiae AH109 (fungal tester) were resuspendedin 1 mL R2A broth to an OD600 of 0.2, and 40 μL of this was mixed with40 mL of warm R2A agar for pouring a single rectangular Petri dish. Seedderived bacteria were inoculated onto plates using a flame sterilized 96pin plate replicator, incubated for 3 days at 28° C. Antibiosis wasscored by observing clear halos around endophyte colonies.

A total of 59 and 72 isolates showed antibiosis activity against eitherE. coli or yeast, respectively (Table 3). Antibiotic production bybacteria on a plant surface or inside a plant may both show that amicrobe is ecologically aggressive (a survivor) and it may mean that itcan help protect a plant against pathogens. Interestingly, three groupsof bacteria, the Bacilli, Enterobacters and Burkholderia both had alarge proportion of isolates (up to 45%, 50% and 88% respectively) whichwere inhibiting growth of E. coli and yeast, suggestive of a commonmechanism of antiobiosis such as production and secretion of a broadspectrum antibiotic. As antibiosis effects were detected in the same 14strains of Burkholderia that produced siderophores, Burkholderiamediated antibiosis may have been be caused by localized ironstarvation, inhibiting both yeast and E. coli growth. A large number ofOchrobacterum isolates also had antagonism towards yeast growth.

Example 4 Seed Endophyte Establishment and Persistence in Corn and Wheat

Seed endophytes colonize plant tissues and as part of their life cyclethey can establish inside roots and disperse systemically throughout theplant vascular system and colonize stems, leaves, flowers and seeds. Inorder to track the fate of individual strains they are labeled with amarker such as Green Fluorescent Proteins (GFP) encoded in a multi copyplasmid. A strain is transformed with the plasmid encoding theexpression of GFP that can be detected by flow cytometry with excitationwith a blue laser at 488 nm and light emission at 530 nm or fluorescentmicroscopy. The transformed strain will fluoresce green and thus can bereadily discriminated from the native microbial community as indigenousgreen fluorescence does not occur in seed endophytes or microbialspecies associated with the rhizosphere or soils. Seeds are inoculatedwith such bacteria which colonize the germinating seed allowing theestablishment, detection and enumeration of the GFP-labeled strain inspecific tissues such as roots, stems and flowers as the plants developand mature. Through the plant's life cycle and reproductive stages thetissues can be analyzed for the presence of the GFP labeled seed-originendophyte. This demonstrates that bacteria's ability to colonize andpersist in vegetative plant tissues, in addition to seed surfaces andinteriors where it was originally inoculated. Seed endophytes will becapable of propagation outside the seed and to be re-established onseeds to colonize new plant generations.

A strain of Pantoea representing OTU#7 and an Enterobacter representingOTU#56 were successfully electroporated with the broad gram negativehost range plasmid, pDSK-GFPuv [Wang et al. (2007), New Phytol 174(1):212-23, incorporated herein by reference]. This is a low copy plasmid,driving constitutive expression of very bright fluorescing GFP under UVlight, in addition to carrying a constitutively expressed kanamycinresistance gene which can allow for selection against background,non-tagged microbes inherent in plant samples. These pDSK-GFPuvtransformed bacteria were grown overnight in a volume of 10 mL of 50%TSB and the next day, CFUs were counted by serial dilution and platingon 50% TSA plates. At this time, 10 g of 58PM36 seed (Blue River Hybridmaize) in a sterile 50 mL conical tube was flooded with a mixture of 10μl of plantability polymer Flo Rite 1706 and 500 μl of the GFP plasmidcontaining OTU#7 or OTU#56 bacteria in R2A broth. After vigorous shakingto ensure even coating of seed with bacteria, tubes were sealed and leftat 25° C. for 7 days, at which time CFUs of bacteria still surviving onseed were assessed by carbide bead beating with a Fastprep24 machine for60 seconds at 5M/seconds. Each 15 mL Falcon tube contained 3 seeds, 2beads and 1 mL of sterile R2A broth in the. After agitation, 20 μL ofthe supernatant was then serially diluted, and 50 μL of the 10× dilutedand 50 μL of the 1,000× diluted plated on halves of 50% TSA plates. Twoof each seed type including untreated, OTU#7-GFP and OTU#56-GFPinoculated seeds were then planted 3 cm deep in 70% ethanol cleaned potscontaining heat sterilized quartz sand, and watered daily withautoclaved water for 7 days as seedlings developed. At this time,seedlings were removed and shaken free from sand, cut into roots orshoots, weighed, placed in 15 mL Falcon tubes along with two carbidebeads and either 1 mL of 50% TSB for shoots or 2 mL of 50% TSB forroots. These were then homogenized by shaking on the Fastprep24 for 120seconds at 5M/second. 20 μL of shoot and root homogenates were thenserially diluted, and 50 μL of the 10× diluted and 50 μL of the 1,000×diluted plated on halves of 50% TSA plates. Uninoculated seed wereplated on antibiotic free TSA, but OTU#7-GFP and OTU#56-GFP plantextracts were placed on TSA plates containing 50 μg/ml of kanamycin. SeeFIG. 1B for an example of the two GFP fluorescing strains on kanamycincontaining TSA plates.

Based on colony counting of serial dilutions, OTU#7-GFP inoculum was ata level of 2.74×10⁹ CFU/mL (approximately 5.08×10⁷ CFU/seed) whenapplied to seeds, and after 7 days at room temperature each seed stillhad about 4.44×10⁵ CFUs per seed. After 7 days of growth in a greenhouseexposed to fluctuations in light, heat, moisture and atmosphere,OTU#7-GFP inoculated seeds developed into a seedling with an average of1.24×10⁶ CFU/g of root tissue and 7.93×10⁵ CFU/g of shoot tissue. Thusafter planting seeds with approximately 4.44×10⁵ CFU of OTU#7-GFP each,seedlings germinated and grew into plantlets containing an average of1.02×10⁶ CFU GFP labelled bacteria. This represents an almost three foldincrease of bacterial numbers and suggests active growth andcolonization of these bacteria in the plant, rather than passivesurvival for a week until the time of harvest.

OTU#56-GFP inoculum was at a level of 1.69×10⁹ CFU/mL (approximately3.13×10⁷ CFU/seed) when applied to seeds, and 7 days later each seedstill had about 2.21×10⁶ CFUs living on its surface. After 7 days ofgrowth in a greenhouse exposed to fluctuations in light, heat, moistureand atmosphere, OTU#56-GFP inoculated seeds developed into seedlingswith an average of 4.71×10⁶ CFU/g of root tissue and 2.03×10⁴ CFU/g ofshoot tissue. Thus after planting seeds with approximately 2.21×10⁶ CFUof OTU#7-GFP each, seedlings germinated and grew into plantletscontaining an average of 6.06×10⁵ CFU GFP labelled bacteria.

Taken together, these two experiments successfully showed that seedderived endophytes are able to survive on a maize seed surface in largenumbers under non-sterile greenhouse conditions for at least a week andare able to colonize and persist on the developing plant over time wherethey will have ongoing opportunities to influence and improve plantgrowth, health and productivity.

Example 5 Colonization of Grass Plants

The establishment of plant-microbe interactions is contingent on closeproximity. The microbiome of the host plant consists of microorganismsinside tissues as well as those living on the surface and surroundingrhizosphere. The present invention describes, among other methods, thecolonization of the plant by application of endophytic microbes of theseed surface. The experiments described in this section are aimed atconfirming successful colonization of plants by endophytic bacteria bydirect recovery of viable colonies from various tissues of theinoculated plant. The experiments were designed to reduce backgroundmicrobes by the use of surface-sterilized seeds, and planting andgrowing the seeds in a sterile environment, to improve the observablecolonization of the plant with the inoculated bacterium.

Experimental Description

Corn seeds of cultivar 58PM36 (Blue River Hybrid) weresurface-sterilizing by exposing them to chlorine gas overnight, usingthe methods described elsewhere. Sterile seeds were then inoculated withsubmerged in 0.5 OD overnight cultures [Tryptic Soy Broth] of strainsSYM00254 (a Micrococcus sp. of OTU 59), SYM00284 (a Pantoea sp. of OTU0), SYM00290 (an Actinobacter of OTU 154), or SYM00292 (a Paenibacillussp. of OTU 6) and allowed to briefly air dry. The seeds were then placedin tubes filled partially with a sterile sand-vermiculite mixture [(1:1wt/wt)] and covered with 1 inch of the mixture, watered with sterilewater, sealed and incubated in a greenhouse for 7 days. After thisincubation time, various tissues of the grown plants were harvested andused as donors to isolate bacteria by placing tissue section in ahomogenizer [TSB 20%] and mechanical mixing. The slurry was thenserially diluted in 10-fold steps to 10⁻³ and dilutions 1 through 10⁻³were plated on TSA 20% plates (1.3% agar). Plates were incubatedovernight and pictures were taken of the resulting plates as well ascolony counts for CFUs.

Experimental Results

Successful inoculation of corn plants by the endophytic bacteria allowedthe recovery of viable, culturable cells as identified on TSA agarplates. Controls experiments using uninoculated, surface sterilizedseeds were conducted and showed few, if any, bacterial cells werecultivatable from the inside suggesting inoculation with extra microbeswould be easily detectable by culturing. Non surface sterilized seedsmeanwhile showed a large diversity of colony types including bothbacteria and fungi which drowned out the detection by culturing ofinoculated bacteria, whereas the plants grown from surface-sterilizedseeds showed a dominance of the inoculated strains readily identified bythe colony morphology.

Finally, significant quantities of viable colonies were recovered fromroots, shoots or seeds of corn plants inoculated with SYM00254,SYM00284, SYM00290, or SYM00292 (Table 10, FIG. 1A), confirming thesuccessful colonization of these tissues of corn plants inoculated withthe various strains. Microbes living on the seed surface can beeliminated by surface sterilization as was done here. The elimination ofthis background allows for the quantitation of the cells of interest.

TABLE 10 Confirmed colonization of seed origin strains in corn shoot androot tissue at 7 days after seed inoculation. Seed-origin microbes Shoottissue Root tissue SYM00254 ++ +++ SYM00284 +++ +++ SYM00290 + +++SYM00292 ++ +++ + — <10⁴ cells per tissue type; ++ — 10⁴ to 10⁶ cellsper tissue type; +++ — >10⁶ cells per tissue type.

Example 6 Testing of Seed-Origin Bacterial Endophyte Populations onPlants

The results shown above demonstrate that many of the endophytic bacteriadescribed herein possess activities that could impart beneficial traitsto a plant upon colonization. First, many of the bacteria described hereare capable of producing compounds that could be beneficial to theplant, as detected using the in vitro assays described above. Inaddition, several representative bacteria were tested and found tosuccessfully colonize corn plants as demonstrated in the example above.The aim of the experiments in this section addresses the ability of thebacterial endophytes to confer beneficial traits on a host plant.Several different methods were used to ascertain this. First, plantsinoculated with bacteria were tested under conditions without any stressto determine whether the microbe confers an increase in vigor. Second,endophyte-inoculated plants were tested under specific stress conditions(e.g., salt stress, heat stress, drought stress, and combinationsthereof) to test whether the bacteria confer an increase in tolerance tothese stresses. These growth tests were performed using three differentmeans: using growth assays on water-agar plates; using growth assays onsterile filter papers; and growth assays on magenta boxes.

Experimental Description

Surface sterilization of seeds—Un-treated organic maize seeds (BlueRiver hybrids, 40R73) and wheat seeds (Briggs, developed by South DakotaUniversity) were sterilized overnight with chlorine gas as follows: 200g of seeds were weighed and placed in a 250 mL glass bottle. The openedbottle and its cap were placed in a dessicator jar in a fume hood. Abeaker containing 100 mL of commercial bleach (8.25% sodiumhypochlorite) was placed in the dessicator jar. Immediately prior tosealing the jar, 3 mL of concentrated hydrochloric acid (34-37.5%) wascarefully added to the bleach. The sterilization was left to proceed for18-24 h. After sterilization, the bottle was closed with its sterilizedcap, and reopened in a sterile flow hood. The opened bottle was left inthe sterile hood for a couple hours to air out the seeds and removechlorine gas leftover. The bottle was then closed and the seeds storedat room temperature in the dark until use.

Seedling Vigor Assessment in Normal and Stressed Conditions on WaterAgar

Bacterial endophytes isolated from seeds as described herein were testedfor their ability to promote plant growth under normal and stressedconditions by inoculating maize and wheat seeds with those endophytesand germinating them on water agar. For each bacterial endophyte tested,5 mL of liquid R2A medium was inoculated with a single colony and theculture grown at room temperature on a shaker to an OD (600 nm) ofbetween 0.8 and 1.2.

Sterilized maize and wheat seeds were placed on water agar plates (1.3%bacto agar) in a laminar flow hood, using forceps previously flamed. Adrop of inoculum with an OD comprised between 0.8 and 1.2 (correspondingto about 10⁸ CFU/mL) was placed on each seed (50 uL for maize, 30 uL forwheat, representing approximately 5·10⁶ and 3·10⁶ CFUs for maize andwheat, respectively). For each treatment, 3 plates were prepared with 12seeds each, arranged as show in on FIG. 2 to insure position uniformity.Plates were sealed with surgical tape, randomized to avoid positioneffects and placed in a growth chamber set at 22° C., 60% relativehumidity, in the dark for four days. After four days, a picture of eachplate was taken and the root length of each seedling was measured usingthe imaging software ImageJ. The percentage difference between thetreated plants and the mock-treated (R2A control) was then calculated.For growth under salt stress, the water agar plates were supplementedwith 100 mM NaCl. For growth under heat stress, the plates were placedat 40° C., 60% humidity after two days of growth, and left for anadditional two days.

Seedling Vigor Assays Under Normal and Stressed Conditions on FilterPaper

Filter papers were autoclaved and placed into Petri dishes, and thenpresoaked with treatment solutions. To simulate normal conditions, 3-4mL sterile water was added to the filters. Drought and saline stresseswere induced by adding 3-4 mL 8% PEG 6000 solution or 50 or 100 mM NaClto the filter papers. Surface sterilized seeds were incubated inbacterial inocula for at least one hour prior to plating. Nine seedswere plated in triplicate for each condition tested, including roomtemperature and heat stress (40° C.) for both normal and salineconditions. During initial stages of the experiment, plates were sealedwith parafilm to inhibit evaporative water loss and premature drying ofthe filter papers. Plates were incubated in the dark at room temperaturefor two days following which heat treatment plates were shifted to 40°C. for 4-6 days. Parafilm was removed from all plates after 3-5 days.After 5-8 days, seedlings were scored by manually measuring root lengthfor corn and shoot length for wheat and recording the mass of pooledseedlings from individual replicates.

Experimental Results

Plant vigor and improved stress resilience are important components ofproviding fitness to a plant in an agricultural setting. These can bemeasured in germination assays to test the improvement on the plantphenotype as conferred by microbial inoculation. The collection ofseed-origin endophytes produced a measurable response in corn (Tables 4aand 4b), and wheat (Table 5a and Table 5b) when inoculated as comparedto non-inoculated controls. For example, from 48 bacterial strains,representing 44 OTUs tested in these germination assays, only 2 did notproduce a favorable phenotype in any of the measured multiple parameterssuch as root length, weight, or shoot length in wheat. This suggeststhat the strains play an intimate role modulating and improving plantvigor and conferring stress resilience to the host plant. In wheat undernormal conditions (vigor), 73% of the strains tested showed some levelof effect and 43% a strong plant response suggesting the physiology andecological niches of the strain collection can be associated to abeneficial plant role. The stress responses in the strain collection canbe seen by the ability of a subgroup to confer a beneficial responseunder different conditions such as heat and salt and drought. These canbe applicable to products for arid and marginal lands. In a largeproportion of cases for the tested strains, the beneficial effect wasmeasurable in both crops indicating that the strains are capable ofcolonizing multiple varieties and plant species. This can play a role intheir mechanisms for dispersal and colonization from one seed into amature plant but also as part of the life cycle to establish an ampledistribution range and ecological persistence in nature. This maytranslate also into relevant features in agriculture. For droughtresponses in corn it was found that 73% of the strains were improving inthe filter paper assay as measured by root length and weight. In somecases it was possible to see additive effects for stress responsescomparing heat, salt and the combination of heat and salt in the sameassay, however not always in a cumulative benefit. For vigor in corn 81%of the strains showed improvements when tested in filter paper or wateragar assays.

The phenotypes conferred by the inoculation and improvement in plantdevelopment are visible by comparing for example the root length, shootlength and weight of the seedling with non-inoculated controls asillustrated by FIGS. 3, 4, 5, and 6.

Individual tests for stress response for corn showed in average 57% ofthe strains an increase in weight over control in heat and salt, 51% forheat-salt and 40% for drought on weight gain. For wheat under saltconditions 54% of the strains produced an effect on root length, 77% ofthe strains a shoot length effect and 50% a weight gain. Drought testswere scored for shoot length and weight with a 59% of the strainsshowing increase in shoot length and 43% weight increase.

Table 4. Systematic assessment of effects of seed-origin microbes oncorn seed vigor under normal and stressed conditions. Legend: “-”indicates no significant increase relative to uninoculated control;“1”=0-5% increase relative to uninoculated control; “2”=5-10% increaserelative to uninoculated control; “3”=>10% increase relative touninoculated control

TABLE 4(a) Assay for seedling vigor in water agar conditions Corncultivar A Corn-organic Weight Root length Root Length Strain OTU#Normal Normal normal salt SYM00002 66 2 2 SYM00011 2 — 1 SYM00012 55 2 2SYM00017c 45 1 3 — SYM00028 18 2 SYM00049 7 2 1 3 1 SYM00052 18 1 —SYM00057b 37 3 2 SYM00060 67 1 SYM00064a 10 2 2 SYM00071 76 1 SYM0007539 2 — — SYM00090 62 — 1 SYM00167 3 1 1 SYM00188 6 1 3 — SYM00192 19 1 2SYM00199 135 1 1 SYM00231 46 2 —

TABLE 4(b) Assay for seedling vigor on filter paper. ROOT LENGTHSEEDLING WEIGHT Corn organic Filter paper Corn organic Filter paperheat- heat- Strain OTU # normal heat salt salt drought normal heat saltsalt drought SYM00002 66 1 3 — — 3 2 3 1 — 1 SYM00011 2 2 — — — — 2SYM00012 55 — 1 — — — 2 2 — 2 — SYM00017c 45 1 — 3 2 2 — 1 1 2 —SYM00028 18 — — — — 3 1 — 2 3 — SYM00033 0 — 1 3 2 2 1 3 — 2 — SYM000497 1 3 1 2 1 — — — 1 — SYM00052 18 2 — — — — 1 SYM00057b 37 1 1 — 1 1 1 31 1 1 SYM00071 76 — 1 2 3 — 2 1 2 3 — SYM00075 39 — — — — — 3 SYM0009062 2 2 2 — 1 3 3 1 1 — SYM00102 38 — 2 3 3 — — 1 — 3 — SYM00107 59 — 1 —— — 1 — — 3 1 SYM00167 3 2 2 1 3 1 1 3 — 2 — SYM00172 146 — — — 1 — — —— — — SYM00188 6 — 1 2 — — 1 2 1 3 — SYM00192 19 — 2 — 3 — 1 2 1 3 —SYM00199 135 — 3 — 3 — 1 3 1 3 — SYM00218 41 — — — 1 — 3 SYM00231 46 — —— — — 1 SYM00508 196 — — — — — 1 — — — — SYM00547 13 2 1 3 — 1 1 — — — 1SYM00554 53 — 3 — 3 — — 2 — 3 — SYM00589 31 — 2 3 3 — 1 3 1 3 — SYM0059512 1 3 2 2 — 1 3 1 3 — SYM00596 9 1 3 3 3 1 — 3 — 3 — SYM00660 1 — 2 1 12 — 2 — — 2 SYM00713 84 — — — — 2 — — — — — SYM00775 30 — — 3 — — 2 2 —3 2 SYM00940 83 — — — — 1 1 1 — — 1 SYM00967 8 — — 3 — 3 1 1 1 — 1SYM00975 51 2 — 3 — 3 1 1 — — 2 SYM00991 36 — — — 3 — 1 — — — 1 SYM00992126 1 — — — 3 — — — — —

Table 5. Wheat Stress/Vigor Test

TABLE 5(a) Wheat seedling vigor assessment using water agar assay. RootLength Wheat Briggs Water-agar Strain OTU# Normal Heat Salt SYM00002 663 — 3 SYM00011 2 3 3 3 SYM00012 55 3 1 3 SYM00015 29 — 1 — SYM00016b 252 3 3 SYM00017c 45 3 2 3 SYM00021 29 3 — — SYM00028 18 3 — 2 SYM00033 03 — 3 SYM00046 56 3 SYM00049 7 3 2 2 SYM00052 18 1 — 3 SYM00057b 37 3 33 SYM00060 67 2 SYM00063 134 1 — — SYM00064a 10 3 — — SYM00071 76 3 — —SYM00075 39 3 — — SYM00090 62 3 2 1 SYM00102 38 2 — — SYM00107 59 2 3 —SYM00167 3 3 — 3 SYM00168 45 3 — 1 SYM00183 10 3 — — SYM00188 6 1 — —SYM00192 19 3 1 — SYM00199 135 3 1 3 SYM00218 41 3 1 — SYM00508 196 3 31 SYM00538A 172 1 — 1 SYM00547 13 2 3 2 SYM00589 31 — 3 1 SYM00595 12 —3 — SYM00596 9 1 3 1 SYM00660 1 — — 2 SYM00713 84 2 — 1 SYM00775 30 — 2— SYM00940 83 — 1 — SYM00965 82 2 — 1 SYM00967 8 2 3 3 SYM00975 51 1 — 2SYM00992 126 — — 3 Legend: “—” indicates no significant increaserelative to uninoculated control; “1” = 0-5% increase relative touninoculated control; “2” = 5-10% increase relative to uninoculatedcontrol; “3” = >10% increase relative to uninoculated control

TABLE 5(b) Wheat seedling vigor using filter paper assay. WHEAT BRIGGSFILTER PAPER Shoot Length Weight Strain OTU # Normal Salt Drought NormalSalt Drought SYM00002 66 — 1 — — 2 — SYM00011 2 3 1 3 3 — 2 SYM00012 55— 2 3 2 — 1 SYM00016b 25 SYM00017c 45 — 1 — — 1 2 SYM00028 18 — 3 3 — 33 SYM00033 0 3 1 2 — — 1 SYM00049 7 3 — 3 2 — 2 SYM00052 18 1 — 1 3 — —SYM00057b 37 3 3 1 2 — 3 SYM00064a 10 — 2 2 — — — SYM00071 76 2 3 3 — 31 SYM00075 39 — 1 3 — — 3 SYM00090 62 — — 3 — — 3 SYM00102 38 — 3 3 2 3— SYM00107 59 1 3 3 2 3 3 SYM00167 3 2 2 1 — — 2 SYM00168 45 SYM00172146 — 3 SYM00188 6 1 3 — — 3 — SYM00192 19 — 3 — 2 3 — SYM00199 135 — —1 2 — — SYM00218 41 — 2 3 3 — 3 SYM00231 46 — — 3 3 3 3 SYM00508 196 — 3— — 2 — SYM00538A 172 SYM00547 13 1 — SYM00554 53 — 3 — — 3 — SYM0058931 — — — — — — SYM00595 12 1 3 3 2 3 — SYM00596 9 1 3 3 1 3 2 SYM00660 13 — SYM00713 84 1 — SYM00965 82 SYM00967 8 — — SYM00975 51 — — SYM00992126 — — Legend: “—” indicates no increase relative to uninoculatedcontrol; “1” = 0-5% increase; “2” = 5-10% increase; “3” = >10% increase

Growth Test of Inoculated Plants in Magenta Boxes

Representative endophytes isolated from seeds as described herein weretested for their ability to promote plant growth under normal andstressed conditions by inoculating maize seeds with those endophytes andgrowing them inside Conviron Growth chambers (Conviron Corp., Asheville,N.C.) on double-decker Magenta boxes essentially as described inRodriguez et al. ISME Journal (2008) 2, 404-416, which is incorporatedherein by reference in its entirety. Briefly, the double-deckers weremade by drilling a hole 8 mm in diameter in the center of a GA-7 plantculture vessel (Magenta boxes, Sigma, St. Louis), top-knotting andweaving through a 14 cm length of cotton rope to the bottom chamber toact as a wick and adding a defined amount of playground sand in theupper chamber. Peter's 20:20:20 plant nutrient solution (PetersFertilizer Co., Fogelsville, Pa.) is added to the bottom chamber and atight-fitting lid is added to the top and the whole system autoclavedand sterilized prior to planting with not-inoculated orendophyte-treated seeds.

Maize seeds were surface sterilized with chlorine gas as describedherein. Sterilized maize seeds were soaked for one hour on theappropriate bacterial culture before planting. Each bacterial culturewas grown on a shaking incubator 20% Tryptic soy broth (TSB) untilreaching ˜0.5 optical density, measured at 600 nm wavelength.Non-inoculated controls were soaked on sterile 20% TSB. Three seeds wereplanted on each double-decker Magenta box and three boxes were used pertreatment (endophytic bacteria×environmental condition). Thedouble-deckers were placed inside a Conviron Growth chamber with asetting of 60% humidity and kept in the dark for four days, until theystarted germinating. Upon germination, plants were grown in a cycle oflight (˜400 mE×m̂−2×ŝ−1) for 14 hrs. and dark for 10 hrs. When the leaveswere fully expanded, approximately 8 days after seeding, the plants wereassigned to one of 3 chambers were conditions were as follows: forControl conditions, plants were kept at 22° C.; for cold, plants weresubjected to 5° C. during the light part of the daily cycle and nearzero degrees during the dark part; for drought, the plants weremaintained in the control chamber, but the liquid from the lower part ofthe double decker was emptied and the soil was allowed to dry; for heatconditions, the light intensity was set to a maximum of ˜600 mE×m̂−2×ŝ−1,while the temperature was set to 40° C. for 12 hrs. out of the 14 hrs.of light and 45 degrees during the two hrs. around noon, during the darkcycle the temperature was set to 30° C. The air humidity was maintainedat 60% in all chambers. The conditions were maintained for one week atthe end of which conductance was measured using an SC-1 Leaf Porometer(Decagon Devices Inc., Pullman, Wash.) in the plants maintained undercontrol and drought conditions and all the plants were harvested,photographed and dried in a convention oven at 45° C. to estimate driedbiomass. Shoot and root lengths were measured digitally using thesoftware ImageJ version 1.48u4 (Rasband http://imagej.nih.gov).

Average measurements were compared against those for uninoculatedcontrols for each treatment. The results obtained with the water agarassay are summarized in Table 7. Several bacterial endophytes providedsignificant plant growth improvement under normal and/or stressedconditions in maize. Notably, strain SYM90 provided growth improvementunder normal, drought and cold conditions, mainly in the form ofincreased root length. Strains SYM00183, SYM00015, SYM00167 and SYM00168also increased root length under drought conditions relative tonon-inoculated controls. Almost all the endophytic bacteria testedprovided increase gain in biomass under cold conditions. The magnitudeof the difference in the conductance between normal conditions anddrought conditions was significantly larger in the plants inoculatedwith SYM231 relative to the non-inoculated controls, suggesting animproved water balance potentially related to closure of stomata.

TABLE 7 Summary of results of testing synthetic combinations ofseed-origin endophytes and corn in plant growth tests on Magenta boxes.Plant vigor and stress resilience in Corn Root length Strain OTU# normaldrought cold SYM00090 62 2 3 3 SYM00016b 25 — — — SYM00231 46 — 2 1SYM00183 10 3 3 2 SYM00015 29 3 3 — SYM00167 3 2 2 — SYM00168 45 2 3 1Legend: “—” indicates no significant increase relative to uninoculatedcontrol; “1” = 0-5% increase relative to uninoculated control; “2” =5-10% increase relative to uninoculated control; “3” = >10% increaserelative to uninoculated control.

Dose Response

Initial experiments described above were conducted to determine whetherthe microbe conferred beneficial traits to the colonized plant. We nextsought to determine the amount of the microbe that is effective toconfer any such benefit. In this example, selected microbial cultureswere diluted to OD₆₀₀ of 1.0, 0.1 and 0.01 (approximately 10⁸, 10⁷, 10⁶CFUs/mL respectively) and applied onto wheat seeds (Briggs) using thewater agar assay previously described.

SYM00011, SYM00033 and SYM00057B cultures were grown from a singlecolony in 5 mL of liquid R2A medium at room temperature on a shaker tostationary phase. The absorbance at 600 nm was measured and adjusted toan OD₆₀₀ of 1.0 (˜10⁸ CFUs/mL) in R2A media. Two additional dilutions atOD 0.1 and 0.01 (˜10⁷ and 10⁶ CFUs/mL respectively) were prepared bydiluting the initial inoculum 10 and 100 times, again in R2A media.

Wheat seeds (Briggs) were sterilized overnight with chlorine gas andplaced on water agar plates as described above. A 30 μL drop of inoculumwas placed on each seed, representing approximately 3.0×10⁶, 3.0×10⁵ and3.0×10⁴ CFUs per seed for OD1, OD0.1 and OD0.01 inoculums, respectively.For each treatment, 3 plates were prepared with 12 seeds each. Plateswere sealed with surgical tape, randomized to avoid position effects andplaced in a growth chamber set at 22° C., 60% relative humidity, in thedark for four days. After four days, a picture of each plate was takenand the root length of each seedling was measured using the imagingsoftware ImageJ (NIH). The percentage difference between the treatedplants and the mock-treated (R2A control) was then calculated.

All doses of the microbes at different concentration provided anincrease in root length over the mock-treated controls as shown in FIG.7. The optimal dose of microbes to confer a growth benefit to wheatvaried for SYM00011, SYM00033 and SYM00057B. For SYM00011, we observed apositive correlation between the bacterial concentration of the inoculumand the growth benefits conferred to the plant, with ˜3.0×10⁶ CFUs/seed(30 μL of OD₆₀₀ of 1.0) being the most effective bacterial amount with a35% increase in growth. For SYM00057B, plants treated with all threedoses had similar root lengths, with the least concentrated inoculum(3×10⁴ CFUs/seed), being the most effective amount, suggestingsaturation at a lower concentration. Similarly, all three concentrationsof SYM00033 provided similar benefits, also suggesting saturation at3×10⁴ CFU/seed.

Inoculation with Strain Combinations

Microbial species colonizing habitats such as the seed-origin endophyticmicrobes described herein are likely to interact with other microbes aswell as with the host in exchanging carbon, energy and othermetabolites. The electron flow and metabolism may involve multiplespecies for complete transfer, which would support the establishment ofsynergistic communities or assemblages. In certain cases, the beneficialeffect of a single microbial inoculant from seeds can be magnified bythe presence of a second synergistic strain favoring the establishmentand persistence of the binary assemblage and their competence. To createassemblages or combinations of available strains in a collection,several approaches were followed, including:

1. Strains with similar or differing functionalities identified by invitro testing. Plant growth promoting activities encompass multiplemicrobial mechanisms to affect plant physiology. These microbialattributes can be tested in vitro such as auxin production, productionof glycosylhydrolases (such as xylanase, cellulase, pectinase, andchitinase, ACC deaminase activity), mineral phosphate solubilization,siderophore production, nitrogen fixation and antibiosis against plantpathogens among others. By combining strains with similar or differingfunctionalities, the plant benefit are improved as compared to theindividual members.

2. Combinations based on strain origin or co-occurrence. Based on theirisolation from the same seed two strains may form an efficient microbialassemblage that can be provided heterologously to novel hosts.

3. Strains with demonstrated germination vigor and/or stress resilience.Seedling germination assays allow testing plant early development,establishment of the seed endophytes in the plant and quantifiablebeneficial effect in root length, weight or shoot length as compared tonon-inoculated controls and the same strains inoculated as singles.

4. Strains isolated from different hosts that may work synergistically.We isolated seed-origin endophytes from multiple plant hosts. Members ofthis group are capable of showing beneficial effects on inoculatedplants when combined as compared to their individual effects.

5. One member from 3 and one member of 4. Seed endophytes showingincreased plant vigor and stress resilience are combined with novel seedendophyte strains and their synergistic interaction amplifies theindividual responses.

These combinations were tested with the collection of seed endophytesrepresenting the 44 OTUs in vigor and stress resilience assays in corn(Table 6a) and wheat (Table 6b).

Table 6. Assessing the Effects of Creating Synthetic Combinations ofMultiple Seed-Origin Endophytes with Seeds

TABLE 6(a) Corn seedling vigor assessment using filter paper assay. RootLength Weight Heat- Heat- Strain 1 Strain 2 Norm Heat Salt salt DroughtNorm Heat Salt salt Drought SYM11 SYM46 — — — — 1 — 2 — — 1 SYM17c SYM46— — — — 3 — — — — 1 SYM33 SYM46 — — — 3 3 — 2 — — 1 SYM49 SYM46 — — — —— — — — — 2 SYM2 SYM46 — — — — 3 — 1 — — 1 SYM172 SYM46 — — 2 — — — — —— — SYM231 SYM46 — — — — 3 — — 1 — 1 SYM11 SYM50 — — 2 2 3 — 3 — — 2SYM17c SYM50 1 — 1 1 2 — — 1 — 1 SYM33 SYM50 — — — 1 1 — 1 — — 2 SYM49SYM50 — — 1 3 — — — — — — SYM2 SYM50 — 1 — 2 — 2 — — — — SYM172 SYM50 —3 1 3 — — 1 — — — SYM231 SYM50 — 2 1 2 — — 2 — — — SYM17c SYM90 — 3 1 3— 1 — — — 2 SYM17c SYM231 1 3 1 3 1 1 1 — — — SYM231 SYM90 — — — 3 — 3 1— — — SYM11 SYM16b — 2 3 3 1 2 — — — 1 SYM11 SYM90 1 — 3 3 3 1 1 — — 1SYM11 SYM102 — 3 3 3 3 2 1 — — 2 SYM11 SYM188 — 3 1 3 3 — — — — 2 SYM16bSYM90 3 SYM90 SYM102 1 3 3 3 3 3 1 — — 3 SYM102 SYM188 1 2 1 3 — — 1 — —— SYM589 SYM90 1 — — 3 3 — — — — — SYM596 SYM90 2 3 1 3 3 3 — — — —SYM218 SYM90 3 SYM57b SYM90 1 3 — 3 3 2 — — — 1 SYM589 SYM231 3 3 — 3 31 — — — — SYM596 SYM231 1 — 2 3 3 2 3 — — 1 SYM102 SYM231 — — 1 3 3 1 —— — — SYM57b SYM231 2 2 1 3 3 2 — 1 — — Legend: “—” indicates nosignificant increase relative to uninoculated control; “1” = 0-5%increase relative to uninoculated control; “2” = 5-10% increase relativeto uninoculated control; “3” = >10% increase relative to uninoculatedcontrol.

TABLE 6(b) Wheat seedling vigor assessment using filter paper assay.Root Length Shoot Length Weight Strain 1 Strain 2 Normal Heat SaltNormal Salt Drought Normal Salt Drought SYM 175 SYM50 — — — — 3 3 — 3 3SYM63b SYM50 1 — — 2 3 3 2 3 3 SYM63b SYM192 3 — — 2 — — 2 — — SYM2SYM17c 3 — 1 1 3 3 1 3 3 SYM167 SYM17c — — — 2 3 3 2 3 3 SYM188 SYM16b —— — 3 — 3 3 — 3 SYM49 SYM16b 3 — — 3 — 3 3 — 3 SYM57b SYM16b 1 2 — 2 3 32 3 3 SYM57b SYM192 2 — 1 — 3 3 — 3 3 SYM11 SYM46 3 — — SYM17c SYM46 2 —1 SYM33 SYM46 3 — — SYM49 SYM46 1 — — SYM2 SYM46 3 1 — SYM172 SYM46 — —— SYM231 SYM46 1 — — SYM11 SYM50 — — — SYM17c SYM50 — — — SYM33 SYM50 —— 1 SYM49 SYM50 3 — — SYM2 SYM50 3 — — SYM172 SYM50 — — 1 SYM231 SYM50 1— 1 SYM965 SYM17c 3 SYM965 SYM90 2 SYM965 SYM231 3 SYM17c SYM90 3 SYM17cSYM231 3 SYM231 SYM90 3 SYM11 SYM16b 2 SYM11 SYM90 3 SYM11 SYM102 3SYM11 SYM188 3 SYM16b SYM90 3 SYM16b SYM102 3 SYM16b SYM188 3 SYM90SYM102 2 SYM90 SYM188 2 SYM102 SYM188 3 SYM2 SYM90 3 SYM589 SYM90 —SYM596 SYM90 1 SYM218 SYM90 3 SYM57b SYM90 3 SYM2 SYM231 1 SYM589 SYM2311 SYM596 SYM231 3 SYM218 SYM231 2 SYM102 SYM231 1 SYM57b SYM231 3Legend: “—” indicates no significant increase relative to uninoculatedcontrol; “1” = 0-5% increase relative to uninoculated control; “2” =5-10% increase relative to uninoculated control; “3” = >10% increaserelative to uninoculated control

A total of 50 binary combinations of strains were tested for vigor andstress resilience for heat, salt, heat-salt or drought and scored forimproved vigor and/or stress resilience. Visible increase in shootlength for wheat was observed when the binaries were paired based ontheir seed origin for isolation and also based on the selection from thevigor and stress resilience assays suggesting that additive effects canbe observed as compared to the effect from individual stresses. Otherinteresting response was observed for corn when inoculated with thebinary formed by SYM00090 related to Chryseobacterium, a representativemember of OTU 62 and and SYM00231 related to Sphingobium and arepresentative member of OTU 46 provided protection against heat-saltstress in corn and vigor in wheat as measured for root length in bothassays. Other combinations with strains was based on the production ofauxin for strains SYM00011, SYM00017c, SYM00033, SYM00049, SYM00002,SYM00062b, SYM00172 and SYM00231 paired with cellulolytic andpetcinolytic from strain SYM00050 showed an enhancement in the heat-saltresilience as compared to the same set of auxin producing strains pairedwith SYM00046 where drought stress was enhanced compared to the previousset. The drought resilience enhancement seen in seedling phenotypes inwheat as compared to controls is the result of the plant response toinoculation and molecular mechanisms for interaction between plant andbacteria. One example is the up-regulation of the protein pectinesterase on inoculated plants and the recognition of that protein indrought protection in plants. In addition, combinations of the 4 strainsSYM00017b, SYM00049, SYM00057b and SYM00188 in corn increaseddramatically the production of the plant hormone Abscisic acid ascompared to individual strains indicating a more beneficial effect atmolecular level with assemblages.

Example 7 Proteomic Analysis of Inoculated Plants

As shown in some of the earlier examples, endophytic microbes describedherein are capable of conferring significant beneficial traits on theinoculated agricultural plant. In order to explore the pathwaysaugmented or otherwise modified by the endophyte, we performed proteomicanalysis on extracts of wheat and corn plants grown on water agar.Sterilized wheat and corn seeds were either mock-inoculated with R2Amedium, or inoculated with selected endophytes SYM00011, SYM00016,SYM00057B, SYM00218, using conditions previously described. The seedswere subjected to the growth parameters as summarized below.

Sample # Crop Test Condition 1 Wheat (Briggs) R2A (mock control) Normal2 Wheat (Briggs) SYM00218 Normal 3 Wheat (Briggs) R2A (mock control)Heat 4 Wheat (Briggs) SYM00011 Heat 5 Wheat (Briggs) SYM00016 Heat 6Wheat (Briggs) SYM00057B Heat 7 Corn (40R73) R2A (mock control) Normal 8Corn (40R73) SYM00057B Normal

Sample Collection:

After 4 days of growth, 12 whole seedlings (including roots, seeds andhypocotyls) per treatment were collected in a 50 mL falcon tube usingsterile forceps and immediately snap-frozen in liquid nitrogen tominimize protein degradation and proteomic changes during samplecollection (such as wound responses from using the forceps). The frozensamples were then homogenized using a pestle and mortar previouslycooled in liquid nitrogen and transferred to a 15 mL falcon tube on dryice. The homogenized samples were stored at −80° C. until furtherprocessing.

Sample Preparation

1 mL of 5% SDS 1 mM DTT was added to 1 mL of homogenized tissue and thesamples were boiled for 5 mins. The samples were cooled on ice and 2 mLof 8M urea solution was added. The samples were spun for 20 mins. at14,000 rpm and the soluble phase recovered. A 25% volume of 100% TCAsolution was added to the soluble phase, left on ice for 20 mins. andcentrifuged for 10 mins. at 14,000 rpm. The protein pellet was washedtwice with ice-cold acetone and solubilized in 125 μL 0.2M NaOH andneutralized with 125 μL of 1M Tris-Cl pH 8.0. Protein solutions werediluted in THE (50 mM Tris-Cl pH8.0, 100 mM NaCl, 1 mM EDTA) buffer.RapiGest SF reagent (Waters Corp., Milford, Mass.) was added to the mixto a final concentration of 0.1% and samples were boiled for 5 min. TCEP(Tris(2-carboxyethyl)phosphine) was added to 1 mM (final concentration)and the samples were incubated at 37° C. for 30 min. Subsequently, thesamples were carboxymethylated with 0.5 mg/ml of iodoacetamide for 30min at 37° C. followed by neutralization with 2 mM TCEP (finalconcentration). Proteins samples prepared as above were digested withtrypsin (trypsin:protein ratio—1:50) overnight at 37° C. RapiGest wasdegraded and removed by treating the samples with 250 mM HCl at 37° C.for 1 h followed by centrifugation at 14,000 rpm for 30 min at 4° C. Thesoluble fraction was then added to a new tube and the peptides wereextracted and desalted using Aspire RP30 desalting columns (ThermoScientific). The trypsinized samples were labeled with isobaric tags(iTRAQ, ABSCIEX, Ross et al 2004), where each sample was labeled with aspecific tag to its peptides.

Mass Spectrometry Analysis

Each set of experiments (samples 1 to 6; samples 7 and 8) was thenpooled and fractionated using high pH reverse phase chromatography(HPRP-Xterra C18 reverse phase, 4.6 mm×10 mm 5 μm particle (Waters)).The chromatography conditions were as follows: the column was heated to37° C. and a linear gradient from 5-35% B (Buffer A-20 mM ammoniumformate pH10 aqueous, Buffer B-20 mM ammonium formate pH10 in 80%ACN-water) was applied for 80 min at 0.5 ml/min flow rate. A total of 30fractions of 0.5 ml volume where collected for LC-MS/MS analysis. Eachof these fractions was analyzed by high-pressure liquid chromatography(HPLC) coupled with tandem mass spectroscopy (LC-MS/MS) using nano-sprayionization. The nanospray ionization experiments were performed using aTripleTof 5600 hybrid mass spectrometer (AB SCIEX Concord, Ontario,Canada)) interfaced with nano-scale reversed-phase HPLC (Tempo, AppliedBiosystems (Life Technologies), CA, USA) using a 10 cm-180 micron IDglass capillary packed with 5 μm C18 Zorbax™ beads (AgilentTechnologies, Santa Clara, Calif.). Peptides were eluted from the C18column into the mass spectrometer using a linear gradient (5-30%) of ACN(Acetonitrile) at a flow rate of 550 μl/min for 100 min. The buffersused to create the ACN gradient were: Buffer A (98% H₂O, 2% ACN, 0.2%formic acid, and 0.005% TFA) and Buffer B (100% ACN, 0.2% formic acid,and 0.005% TFA). MS/MS data were acquired in a data-dependent manner inwhich the MS1 data was acquired for 250 ms at m/z of 400 to 1250 Da andthe MS/MS data was acquired from m/z of 50 to 2,000 Da. For Independentdata acquisition (IDA) parameters MS1-TOF 250 ms, followed by 50 MS2events of 25 ms each. The IDA criteria, over 200 counts threshold,charge state +2-4 with 4s exclusion. Finally, the collected data wereanalyzed using Protein Pilot 4.0 (AB SCIEX) for peptide identificationsand quantification.

Results:

The proteomics analysis of wheat inoculated with endophytic bacteria(SYM11, SYM16B and SYM57B) grown under heat stress and maize inoculatedwith SYM57B grown under normal condition revealed three major pathwaysaugmented or otherwise modified by the endophyte: growth promotion,resistance against oxidative stress and mechanisms involved in symbiosisenhancement (Table 8 and Table 9).

TABLE 8 Proteins showing differential levels of expression under heatstress in endophyte-inoculated wheat (var. Briggs) seedlings relative tonot-inoculated control seedlings. UP-REGULATED PROTEINS IN RESPONSE TOENDOPHYTIC BACTERIA Ratio Treatment/Control Accession number Gene namePathway SYM-00011 SYM-00016B SYM-00057B Growth promotion gi|474293349Acid beta-fructofuranosidase mobilization of sucrose 0.5-1 Fold 1-2 Fold1-2 Fold gi|473798701 ATP synthase subunit beta, ATP synthesis 1-2 Fold1-2 Fold mitochondrial gi|473945263 Fructan 1-exohydrolase mobilizationof fructans 1-2 Fold gi|473798921 Glutamine synthetase cytosolic Aminoacid biosynthesis 1-2 Fold 1-2 Fold isozyme 1-2 gi|474427549Dynamin-related protein 1E Cell division 1-2 Fold 1-2 Fold 1-2 Foldgi|474154210 Histone H1 Cell division 1-2 Fold 1-2 Fold 1-2 Foldgi|474396419 Histone H1 Cell division 1-2 Fold 1-2 Fold gi|474315053Histone H2A Cell division 1-2 Fold 1-2 Fold >2 Fold gi|474114390 HistoneH2A Cell division 1-2 Fold gi|474408930 Histone H2A.1 Cell division 1-2Fold >2 Fold gi|474247555 Protein H2A.7 Cell division 1-2 Fold 0.5-1Fold gi|474400621 Histone H4 Cell division 1-2 Fold 1-2 Foldgi|474160133 Serine carboxypeptidase-like protein Amino acid release 1-2Fold 1-2 Fold 1-2 Fold gi|474397165 Serine carboxypeptidase-like 51Amino acid release >2 Fold 1-2 Fold gi|474449933 Pectinesterase 1 Cellwall remodeling 1-2 Fold >2 Fold gi|474193958 Peptidyl-prolyl cis-transisomerase Juvenile phase of 1-2 Fold >2 Fold >2 Fold CYP40 vegetativedevelopment gi|473956589 Ribonucleoside-diphosphate DNA synthesis0.1-0.5 Fold 0.1-0.5 Fold >10 Fold reductase gi|474326915 Villin-4 Cellelongation >2 Fold >10 Fold >2 Fold gi|474156626 Glutenin, low molecularweight Protein storage - affected 1-2 Fold 1-2 Fold subunit by heatResistance against abiotic stress gi|474449933 Pectinesterase 1Resistance to drought 1-2 Fold >2 Fold gi|474381202 Peroxiredoxin Q,chloroplastic Resistance to oxidative 0.5-1 Fold 0.5-1 Fold >2 Foldstress gi|474299547 Glutathione S-transferase DHAR3, Resistance tooxidative 1-2 Fold 1-2 Fold >2 Fold chloroplastic stress gi|474276683Peroxidase 12 Resistance to oxidative 1-2 Fold 1-2 Fold 1-2 Fold stressgi|474414579 3-hydroxybenzoate 6-hydroxylase 1 Degradation of toxic 1-2Fold >2 Fold 1-2 Fold organic compounds gi|474323467 BAHDacyltransferase DCR Cutin formation - 1-2 Fold 1-2 Fold 0.1-0.5 Folddessication resistance gi|473999626 5′-methylthioadenosine/S- Negativefeedback on 0.5-1 Fold 0.5-1 Fold 0.5-1 Fold adenosylhomocysteinenucleosidase ethylene production gi|474326305 Aldehyde dehydrogenasefamily 2 Controls acetaldehyde 0.5-1 Fold 0.5-1 Fold 0.5-1 Fold memberC4 accumulation gi|474041937 putative protein phosphatase 2C 45Regulates ABA signaling 0.5-1 Fold gi|473894812 DEAD-box ATP-dependentRNA mRNA decay and 0.1-0.5 Fold helicase 40 (“DEAD” disclosed asribosome biogenesis SEQ ID NO: 1449) Symbiosis enhancement gi|474407144Enolase 1 Glycolisis of sugars 0.5-1 Fold 0.5-1 Fold required byendophyte gi|474119301 Protochlorophyllide reductase B, Affected bysymbiosis 0.5-1 Fold chloroplastic gi|474213532 Elicitor-responsiveprotein 1 Microbe response 0.5-1 Fold 0.5-1 Fold 1-2 Fold signaling

TABLE 9 Proteins showing differential levels of expression under normalcondition in endophyte-inoculated corn (40R73) seedlings relative tonot-inoculated control seedlings. Growth promotion Accession SYM- numberGene name Pathway 00057B/control gi|413950290 putative peptidyl-prolylcis-trans Organ development >2-fold isomerase gi|414876902 ATP-dependentClp protease Chloroplast component >2-fold proteolytic subunitgi|413948820 Translation elongation factor Tu Protein biosynthesis 1-2fold isoform 3 gi|414878150 Chaperone protein dnaJ 15 Positivegravitropism <0.5-fold gi|413954599 translation elongation/initiationEmbryo development ends <0.5-fold factor seed dormancy Accession SYM-number Gene name Function 00057B/control Resistance against abioticstress gi|414867473 Glutathione S-transferase GSTU6 Resistance tooxidative 1-2 fold stress gi|414876903 Calmodulin2 ABA-inducedantioxidant <0.5-fold defense gi|413920116 Ras protein Rab-18 ABAinducible, accumulates 0.5-1 fold in cold stress gi|413926351 DNA repairprotein RAD23-1 isoform 3 Nucleotide-excision repair 0.5-1 foldSymbiosis enhancement gi|413920282 Hydroquinone glucosyltransferaseUpregulated in Rhizobia >10-fold symbiosis gi|413939151 replicationfactor C subunit 3 Negative regulation of >10-fold defense responsegi|413946904 NEDD8-activating enzyme E1 catalytic Proteinneddylation - >10-fold subunit microbe response gi|413951445delta3,5-delta2,4-dienoyl-CoA Peroxisome component - >10-fold isomerasedefense gi|413925737 Proteasome subunit alpha type Response tocompatible >2-fold symbiotic bacteria gi|413957021 Ras protein RHN1Legume homolog involved >2-fold in nodulation gi|414875813 Early nodulin20 Root nodule formation >2-fold gi|414886632 Putative plant regulatorRWP-RK Nodule inception protein 1-2 fold family protein gi|413955359putative metacaspase family protein Programmed cell death 0.5-1 foldregulation gi|413920552 win1 Defense response to <0.5-fold bacteria andfungi gi|413948744 protein brittle-1 Response to nematodes <0.5-foldgi|414869634 Proteasome subunit beta type Regulation of 0.5-1 foldhypersensitive response

Growth Promotion:

Proteins involved in the breakdown of seed stored reserves and playingimportant roles in the stimulation of continued growth duringgermination were up-regulated by endophytes. This class of proteinsincludes beta-fructofuranosidases, fructan 1-exohydrolases andcarboxypeptidases involved in the mobilization of sucrose, fructans andinsoluble proteins respectively, for the release of glucose, fructoseand amino acids [Fincher (1989). Annu. Rev. Plant Physiol. Plant Mol.Biol. 40:305-46, incorporated herein by reference in its entirety].Those results show that bacterial endophytes induce a faster release ofnutrients from the seed, leading to augmented growth at early stage ofplant development. The levels of proteins playing a role in cellproliferation and elongation were also increased in endophyte-inoculatedseedlings. This class of proteins includes dynamins, histones, aribonucleoside-diphosphate reductase, pectinesterases and villins,involved in cell division, chromatin structure, DNA synthesis, cell wallremodeling and elongation respectively [Hepler et al. (2001) Annu. Rev.Cell Dev. Biol. 2001. 17:159-87, Kang et al. (2003) The Plant Cell 15:899-913, Imoto et al. (2005) Plant Mol. Biol. 58:177-192, incorporatedherein by reference in their entirety). Those results demonstrate that,in response to the endophytic bacteria tested, the two types of plantgrowth, proliferation and elongation, are promoted, leading tosubstantial growth enhancement.

Resistance Against Stress:

A number of proteins involved in resistance against stress weresignificantly up-regulated in wheat under stress induction and thepresence of endophytes. The level of several proteins playing a role inresistance against oxidative stress by scavenging reactive oxygenspecies was higher in inoculated plants including glutathioneS-transferases (GST), peroxidase and ascorbate oxidase [Apel and Hirt(2004) Annu. Rev. Plant Biol. 55:373-99, incorporated herein byreference in its entirety]. Those results shows that in addition toplant growth, the endophytes tested promoted the general pathwaysinvolved in resistance against oxidative stress. The proteomics data-setalso revealed the strong induction of a pectinesterase by SYM11 andSYM57B in wheat that might play a role in drought resistance aspreviously described (WO2013122471 incorporated herein by reference inits entirety).

Symbiosis Enhancement: In corn under normal conditions, only GST wasup-regulated, while other abscisic acid (ABA) and stress inducibleproteins were down-regulated. The down-regulation of ABA and stressinducible proteins in corn was positively correlated with thedown-regulation of proteins associated to programmed cell death,pathogen resistance and hypersensitive response. Moreover, thereplication factor C, subunit 3 that negatively regulates plant defensewas significantly overexpressed in the SYM57b inoculated corn seedlings.Those results are consistent with the conventional wisdom that, undernormal condition, the establishment of symbioses with beneficialmicrobes involves decrease in the expression of genes associated to theplant defense system [Samac and Graham (2007) Plant Physiol.144:582-587, incorporated herein by reference in its entirety].

In addition, several proteins directly associated with beneficialsymbioses are up-regulated in the wheat and corn. It is intriguing thatseveral of these proteins are homologous to proteins involved in noduleformation in legumes. Many genes involved in nodulation, such asnodulation receptor kinases are broadly distributed in the plantkingdom, even in plants incapable of forming nodules, as is the case ofmaize [Endre et al. (2002) Nature 417:962-966, incorporated herein byreference in its entirety]. Some of these conserved receptors may sensebacterial signals in symbiotic associations other than Legume-Rhizobiaand this may explain why the nodulation factors from Badyrhizobiumjaponicum are able to enhance seed germination and root growth in corn[Suleimanov et al. (2002) J. Exp. Bot. 53:1929-1934, incorporated hereinby reference in its entirety].

Example 8 Analysis of Hormone Levels in Inoculated Plants

As shown in some of the earlier examples, endophytic microbes describedherein are capable of conferring significant beneficial traits on theinoculated agricultural plant. In order to explore the possibility thatseed endosymbionts augment or modify hormone levels in planta, ametabolomic analysis was performed of 12 phytohormones(indole-3-carboxylic acid, trans-zeatin, abscisic acid, phaseic acid,indole-3-acetic acid, indole-3-butyric acid, indole-3-acrylic acid,jasmonic acid, jasmonic acid methyl ester, dihydrophaseic acid,gibberellin A3, salicylic acid) in wheat and corn plants grown on wateragar under normal condition and inoculated by SYM57B or a mix ofselected endophytes (see table below). The mixes of endophytes inoculumswere obtained by mixing equal volume of the different bacterialcultures.

Crop Treatment Wheat (Briggs) R2A (mock control) Wheat (Briggs) SYM57BWheat (Briggs) Mix (SYM11 + SYM17C + SYM49 + SYM57B) Corn (40R73) R2A(mock control) Corn (40R73) SYM57B Corn (40R73) Mix (SYM17C + SYM49 +SYM57B + SYM188)

Samples Analyzed for Plant Hormone Profiling Methods Sample Preparation

4-day old whole wheat and corn seedlings (including roots, seed andhypocotyl) were finely ground in liquid nitrogen by mortar and pestlethen aliquoted into 1.5 mL microcentrifuge tubes and weighed.Phytohormones were extracted from ground sprouts using a proteinprecipitation protocol where cold extraction solvent (80% aqueousmethanol with 1% acetic acid) containing internal standards was added tothe finely ground plant material (400 μL solvent for every 100 mg groundplant tissue). Samples were kept on ice during the addition ofextraction solvent. Samples were then vortexed for 60 min at medium-highspeed at 4° C., then centrifuged for 15 min at 13,000 g at 4° C. Theresultant supernatant was removed and analyzed by LC-MS/MS.

LC-MS/MS

Phytohormones were chromatographically separated using a WatersnanoAcquity UPLC system on a Waters Atlantis dC18 column (3 μM, 300μM×150 mm) held at 40° C. Samples were held at 4° C. in theauto-sampler. Water (buffer A) and acetonitrile (buffer B), both with0.1% formic acid, were used as buffers. The flow rate was 11.5 μL/minand injection volume 1 μL. Each sample was injected twice and hormonelevels averaged. Phytohormones were analyzed by selected reactionmonitoring (SRM) on a Waters Xevo TQ-S mass spectrometer in bothnegative and positive ion modes. The UPLC gradient was as follows: time(t)=0 min, 10% B; t=0.5 min, 10% B; t=5.5 min, 95% B; t=7.5 min, 95% B;t=8 min, 10% B. The column was equilibrated for three minutes beforeeach injection.

Results

Inoculation of wheat and corn with seed-origin endophytes significantlyaltered the level of several plant hormones, includingindole-3-carboxylic acid, trans-zeatin, abscisic acid, phaseic acid andindole-3-acetic acid. In addition, the combination of multiple seedendosymbionts further modified the plant hormone profiling of inoculatedplants. In particular, the level of abscisic acid andindole-3-carboxylic acid, the decarboxylated form of auxin, wasaugmented by 63% and 98% respectively in corn inoculated with the mixedendophytes.

Example 9 Field Trial Planting & Assessment of Plant Health Under Stress

Planting & setup of field trials in normal and stressed conditions

To determine whether a microbe or combination of microbes is capable ofpromoting plant growth in the field, a field trial was conducted usingrepresentative endophytic microbes described herein. The trial involvedtesting individual microbial strains and combinations of strains bytreating and planting the seeds of a variety of plants (including, butnot limited to maize, wheat, cotton, and barley), with one or twovarieties or cultivars of each plant tested. A typical trial was laidout as a randomized complete block design, with each combinationmicrobial treatment and plant variety replicated six times in the trial.

Trials were conducted across various geographies including field sitesin major producing regions of South Dakota, Nebraska, Saskatchewan andAustria, on both dry and irrigated land to test responses in bothwell-watered and drought-stressed conditions. Trials may also beconducted in geographies with hotter growing seasons, where temperaturescan reach up to 95° F. for five or more consecutive days, in order toassess responses under heat stress. Trials may also be conducted ingeographies prone to higher levels of microbial, nematode or insectpathogens in order to assess responses under pathogen stress

Fertilizer and herbicides are applied according to soil test results andlocally recommended practice. Fertilizer may be applied at 25%, 50% or75% of recommended levels to assess responses under nutrient stress.

For maize, typical field plots were 10′×′40′ with 4 evenly spaced rows,seeded at a rate of approximately 34,000 seeds per acre. Each randomizedcomplete block trial included an untreated control and amock-formulation control, as well as additional untreated border plotson the 40′ ends. For wheat, typical field plots were 5′×50′ with 7evenly spaced rows, seeded at a rate of approximately 90 lbs per acre.Each randomized complete block trial included an untreated control and amock-formulation control.

Measurement of Biomass

Biomass of field plots is assessed by selecting 10 plants per plot formaize or 20 plants per plot for wheat at random from the middle two rowsat harvest, removing the plants from the soil and cleaning off anyresidual soil. Plants are then divided into aerial and root sections andweighed to obtain fresh weight. Plants are then be dried in a vacuumoven overnight and weighed again to obtain dry weight.

Measurement of Yield, Grain Moisture, Test Weight

Yield of field plots is measured at the end of the growing season byharvesting the plots with an appropriate harvester. For maize, only themiddle two rows are harvested. For wheat, all 7 rows may be harvested,or only the middle 5 may be used. Test weight and moisture of the grainmay be recorded by the harvester, or subsamples of the harvested grainmay be used for manual test weight assessment and moisture analysis in aDICKEY-John® grain moisture analyzer (Dickey-John Corp., Chatham, Ill.),using parameters recommended by the manufacturer.

Measurement of Emergence & Plant Height

Emergence in the field plots was assessed for wheat by counting thenumber of emerged plants in the middle 10′ section of the middle tworows and reporting the total number plants emerged. Emergence countswere done every four days starting with the day of emergence of thefirst plants and ending when 50% or more of the plants in the plot hadreached Feekes scale 2. Emergence in the field was assessed for maize bydoing a full count of all emerged plants in the plot and reporting thenumber of emerged plants as a percentage of the number of seeds plantedin that plot. Two emergence counts were done, one at the emergence ofthe first plants and a second count five days later.

Emergence of wheat in a field trial on four different days is shown inthe top panel of FIG. 8. The numbers reported are an average ofemergence counts of 6 replicate plots for each treatment. All SYMstrains show improvement in emergence over the untreated control, withSYM00028 showing the greatest improvement.

Emergence of corn in a field trial is shown in the middle panel of FIG.8 (for a dryland trial) and in the bottom panel FIG. 8 (for an irrigatedtrial). The numbers are reported as a percent increase over an untreatedcontrol and were calculated as an average of emergence counts of 6replicate plots for each treatment. All SYM strains show improvement inemergence over the untreated control. SYM00260, SYM00290 and SYM00254showed the best performance in the dryland trial, and SYM00292 showedthe best performance in the irrigated trial.

Measurement of Flowering Time

The day of flowering for a particular plot is recorded when 50% or moreof the plants in the plot have reached the flowering stage.

SPAD Measurement

Chlorophyll values, for example, SPAD readings are conducted on wheat bymeasuring 10 plants per plot at random from the middle two rows. Thefirst measurement is done at flowering, with a second measurement donetwo weeks later on the same 10 plants in each plot. The SPAD reading istaken on the flag leaf on each plant, for example, as measured withSPAD502 supplied by Minolta Co., Ltd., at approximately three quartersof the leaf length from the leaf base and avoiding the midrib of theleaf. SPAD readings are conducted on maize by measuring 10 plants perplot at random from the middle two rows. The first measurement is doneat flowering (VT stage), with a second measurement done two weeks lateron the same 10 plants in each plot. The SPAD reading is taken on thetopmost leaf under the tassel, approximately 0.5 inch from the edge ofthe leaf and three quarters of the leaf length from the leaf base.

Stand Count & Lodging Assessment

Stand count and percent lodging are assessed in wheat by counting thetotal number of tillers and the number of broken stalks in the middletwo rows on the day of harvest. Stand count and percent lodging areassessed in maize by counting the number of standing plants and thenumber of stalks broken below the ear in the middle two rows on the dayof harvest.

Sterilization of Seed Surfaces from Microorganisms Using DisinfectingChemicals

Example Description

In order to isolate and characterize endophytic microorganisms, allmicroorganisms living on the surface of the plant, plant tissue, orplant structure must be removed. After a pre-wash to remove all looselyattached microorganisms, the surfaces are sterilized with disinfectingchemicals, and the plant tissue is tested for sterility.

Experimental Description

Surface sterilization of seeds is performed as described by Bacon andHinton, “Isolation, In Planta Detection, and Uses of Endophytic Bacteriafor Plant Protection”, Chapter in Manual of Environmental Microbiology,3rd Edition (2007): 638-647, with some modifications. Briefly, batchesof seeds are first given a prewash to remove as many of the surfacebacteria as possible by vigorously washing them in sterile 0.05Mphosphate buffer (pH 7.2). If the seeds have been treated withpesticides, the prewash buffer contains 0.01% Tween 20 or 0.05% TritonX-100, and is followed by 3-5 washes in 75-90% ethanol. They then areallowed to imbibe in 1× sterile phosphate-buffered saline (PBS) at 4° C.for 24 h (or different times depending on the seed variety). Afterimbibing, they are surface sterilized by immersion in 70% ethanol for 5min, 10% bleach solution for 3-15 minutes (depending on seed), andrinsed twice with autoclaved distilled water. Alternatively, samples canbe surface-sterilized by using 1% chloramine for 3 minutes followed by70% ethanol for 5 minute and rinsed twice with sterile distilled water.Alternatively, seeds can be sterilized by submerging them in 10%hydrogen peroxide for 5 min-1 hour and then rinsed twice with steriledistilled water. Samples are blotted dry using autoclaved paper towels.Once sterilized, a few seeds of each batch are aseptically imprintedonto Tryptic Soy Agar (TSA) and Potato Dextrose agar (PDA) in a Petridish using sterile forceps: one of the sides of the seed is pressedfirst, then the seed is turned onto its other side on another part ofthe plate, and then removed. These plates are stored in the dark forfive days and checked daily for bacterial and/or fungal growth. If thebatch of seeds proves to retain microbes, the whole batch is destroyedand the experiment re-started with new seeds.

Other plant tissues are surface-sterilized and tested for sterilityessentially as described for seeds, with some modifications:

1. Leaves: Leaves are detached and pre-washed as described for seeds.Then they are placed in 1% chloramine for 30 minutes, or in fullstrength commercial bleach for 5 minutes, and washed twice in 3-5 timesin distilled sterile water for 3 minutes. Samples are blotted dry usingautoclaved paper towels. 5 grams of leaf tissue are then transferred toa sterile blender and processed as described for seeds with 50 mL of R2Abroth.

2. Roots: Roots are removed from the plants and pre-washed twice asdescribed for seeds to remove all attached soil. Roots aresurface-sterilized in 1% chloramine solution for up to 30 minutes (oralternatively 10% bleach) and washed 3-5 times in distilled sterilewater for 3 minutes. The roots are then immersed for 30 minutes insterile 0.05 M phosphate buffer (pH 7.2) and then rinsed several times.5 g of root tissue are then transferred to an sterile blender andprocessed as described for seeds with 50 mL of R2A broth

3. Stems: A portion of the plant stem is cut from the plant pre-washedas described for seeds. It is then surface sterilized as described forthe roots and washed twice in distilled sterile water for 3 minutes. 5 gfrom the inside of the stem are removed by cutting the outside layerwith a sterile blade and processed as described for seeds.

Sterilization of Seed or Plant Surface from Bacteria Using AntibioticAgents

Seeds are surface sterilized with antibacterial compounds such as sodiumhypochlorite, copper oxychloride, copper hydroxide, copper sulfate,chlorothalonil, cuprous oxide, streptomycin, copper ammonium carbonate,copper diammonia diacetate complex, copper octanoate, oxytetracycline,fosetyl-AL or chloropicrin. Seed is soaked in an aqueous solution orcommercial formulation containing one or more of these compounds for 30seconds to 12 hours in a plastic container. The solution may also beadministered to seedlings or plants by spraying or soaking leaves orother aerial parts of the plant, after which the plant tissues aresprayed or rinsed with water to remove residual fungicide. After surfacesterilization, the seed is removed from the antibacterial formulationand washed 3-5 times with sterile distilled water.

Sterilization of Seed or Plant Surface from Fungi Using FungicidalAgents

Seeds are surface sterilized by use of contact fungicides such ascaptan, maneb, thiram, fludioxonil, and others. Seed is soaked in anaqueous solution or commercial formulation containing one or more ofthese compounds for 30 seconds to 12 hours in a plastic container. Aftersurface sterilization, the seed is removed from the fungicide solutionand washed 3-5 times with sterile distilled water. The solution offungicides may also be administered to seedlings or plants by sprayingor soaking leaves or other aerial parts of the plant, after which theplant tissues are sprayed or rinsed with water to remove residualfungicide. Systemic fungicides such as azoxystrobin, carboxin,mefenoxam, metalaxyl, thiabendazole, trifloxystrobin, difenoconazole,ipconazole, tebuconazole or triticonazole may also be used only when itis desirable to also sterilize interior tissues.

Isolation of Bacteria and Fungi from the Interior of Seeds

Example Description

Isolation of fungi and bacteria (including endophytes) from the interiorof surface-sterilized seeds is done using techniques known in the art.Surface sterilized seeds are ground, diluted in liquid media, and thissuspension is used to inoculate solid media plates. These are incubatedunder different conditions at room temperature.

Experiment Description

Approximately fifty surface-sterilized seeds are transferred asepticallyto a sterile blender and ground. The ground seeds are resuspended in 50mL of sterile R2A broth, and incubated for 4 h at room temperature. Ten1 mL aliquots of the seed homogenates are collected and centrifuged,their supernatants discarded and the pellets gently resuspended in 1 mLof sterile 0.05 phosphate buffer; 0.5 mL of 50% glycerol is added toeach of five tubes. These are stored at −80 C for possible furthercharacterization (i.e. if the plates become too overcrowded with onemicroorganism, the frozen aliquots can be used to plate lower dilutionsof the homogenate). The remaining aliquots are diluted down twice inhundred-fold dilutions to 10⁻⁴. 100 microliters of the 1, 10⁻², and 10⁻⁴dilutions are used to inoculate three Petri dishes containing thefollowing media in order to isolate of bacteria and/or fungi:

-   -   1. Tryptic Soy agar    -   2. R2A agar    -   3. Potato dextrose agar    -   4. Sabouraud Agar    -   5. Other media depending on target microorganism

The plates are divided into three sets comprising each media type andincubated in different environments. The first set is incubatedaerobically, the second under anaerobic conditions, and the third undermicroaerophilic conditions and all are inspected daily for up to 5 days.1-2 individual colonies per morphotype are isolated and streaked forpurity onto fresh plates of the same media/environment from which themicroorganism was isolated. Plates are incubated at room temperature for2-5 days. Once an isolate grows it is streaked once more for purity ontoa fresh plate of the same media to ensure purity and incubated under thesame environmental conditions.

From the second streaked plate, isolates are stored in Tryptic soybroth+15% glycerol at −80° C. for further characterization, by firstscraping 2-3 colonies (about 10 μL) from the plate into a cryogenic tubecontaining 1.5 mL of the above-mentioned media and gently resuspendingthe cells. Alternatively, isolates are propagated in specialized mediaas recommended for the particular taxon of microorganism. The microbesobtained represent those that live in the seeds of the plant accession.

Isolation of Bacteria and Fungi from Plant Interior Tissues

Example Description

Isolation of fungi and bacteria (including endophytes) fromsurface-sterilized plant tissues is done using techniques known in theart. Surface sterilized plant tissues are ground, diluted in liquidmedia, and then this suspension is used to inoculate solid media plates.These are incubated under different environmental conditions at roomtemperature.

Experiment Description

Approximately fifty grams of surface-sterilized plant tissue aretransferred aseptically to a sterile blender and ground. The groundtissue is resuspended in 50 mL of sterile R2A broth, and incubated for 4h at room temperature. Ten 1 mL aliquots of the plant tissue homogenatesare collected and centrifuged, their supernatants discarded and thepellets gently resuspended in 1 mL of sterile 0.05 phosphate buffer. 0.5mL of 50% Glycerol is added to each of five tubes. These are stored at−80° C. for possible further characterization (i.e. if the plates becometoo overcrowded with one microorganism, the frozen aliquots can be usedto plate lower dilutions of the homogenate). The remaining aliquots arediluted down twice in hundred-fold dilutions to 10⁻⁴. One hundredmicroliters of the 1, 10⁻², and 10⁻⁴ dilutions are used to inoculatethree Petri dishes containing the following media in order to isolate ofbacteria and/or fungi:

-   -   1. Tryptic Soy agar    -   2. R2A agar    -   3. Potato dextrose agar    -   4. Sabouraud Agar    -   5. Other media depending on target microorganism

Plates are divided into three sets comprising each media type andincubated in different environments. The first set is incubatedaerobically, the second under anaerobic conditions, and the third undermicroaerophilic conditions and all are inspected daily for up to 5 days.1-2 individual colonies per morphotype are isolated and streaked forpurity onto fresh plates of the same media/environment from which themicroorganism was isolated. Plates are incubated at room temperature for2-5 days. Once an isolate grows it is streaked once more for purity ontoa fresh plate of the same media to ensure purity and incubated under thesame environmental conditions.

From the second streaked plate, isolates are stored in Tryptic soybroth+15% glycerol at −80° C. for further characterization, by firstscraping 2-3 colonies (about 10 μL) from the plate into a cryogenic tubecontaining 1.5 mL of the above-mentioned media and gently resuspendingthe cells. Alternatively, isolates are propagated in specialized mediaas recommended for the particular taxon of microorganism. The catalog ofmicrobes thus isolated constitutes a good representation of the microbesfound in the growing plant tissues.

Isolation of Bacteria and Fungi from Plant or Seed Surfaces

To collect phyllosphere, rhizosphere, or spermosphere material forculturing of microbes, unwashed shoot, roots or seeds are shakenfree/cleaned of any attached soil and stuffed into sterile 50 mL Falcontubes. To these, 10 mL of sterile 0.1 M sodium phosphate buffer is addedand shaken, followed by 5 minutes of sonication to dislodge microbesfrom plant surfaces, with the resulting cloudy or muddy wash collectedin a separate 15 mL Falcon tube. 100 μL of this microbe filled wash canbe directly spread onto agar plates or nutrient broth for culturing andenrichment, or it can be further diluted with sterile 0.1 M sodiumphosphate buffer by 10×, 100×, 1,000×, 10,000× and even 100,000×, beforemicrobial culturing on agar plates or nutrient broth. Glycerol stockpreparations of the plant surface wash solution should be made at thispoint by mixing 1 mL of the soil wash solution and 0.5 mL of sterile,80% glycerol, flash freezing the preparation in a cryotube dipped inliquid nitrogen, and storing at −80° C. Nutrient broth inoculated with amixture of plant surface bacteria should form a stable, mixed communityof microbes which can be used in plant inoculation experiments describedherein, subcultured in subsequent broth incubations, or spread on agarplates and separated into individual colonies which can be tested viamethods described herein.

Characterization of Fungal and Bacterial Isolates

Example Description

Characterization of fungi and bacteria isolated from surface-sterilizedor non-sterilized plant or seed tissues is done using techniques knownin the art. These techniques take advantage of differential staining ofmicroorganisms, morphological characteristics of cells, spores, orcolonies, biochemical reactions that provide differentialcharacterization, and DNA amplification and sequencing of diagnosticregions of genes, among other methods.

Experimental Description

Isolates of bacteria and/or fungi isolated as described herein(including endophytic bacteria and fungi) are categorized into threetypes: bacterial isolates, fungal isolates, and unknown isolates (sinceyeast colonies can resemble bacterial colonies in some cases) based oncolony morphology, formation of visible mycelia, and/or formation ofspores. To determine if an unknown isolate is bacterial or fungal,microscopic analysis of the isolates is performed. Some of the analysesknown to the art to differentiate microorganisms include the 10% KOHtest, positive staining with Lactophenol cotton blue, Gram staining, andgrowth on media with selective agents. The distinguishing featuresobserved by these tests are relative cell size (yeast size is muchlarger than bacterial size), formation of hyphae and spores (filamentousbacteria form smaller hyphae than fungi, and do not form structurescontaining spores), or growth under selection agents (most bacteria cangrow in the presence of antifungal compounds like nystatin, while mostfungi cannot; likewise, most fungi are unaffected by the presence ofbroad-spectrum antibiotics like chloramphenicol and spectinomycin).

To identify the isolates, DNA sequence analysis of conserved genomicregions like the ribosomal DNA loci is performed. To obtain DNA toperform PCR amplifications, some cellular growth from solid media(approximately 5-10 μL) is resuspended in 30 μL of sterile Tris/EDTAbuffer (pH 8.0). Samples are heated to 98° C. for 10 minutes followed bycooling down to 4° C. for 1 minute in a thermocycler. This cycle isrepeated twice. Samples are then centrifuged at 13,000 RCF for 1-5minutes and used as DNA template for PCR reactions. Below is a series ofprimer combinations that can be used to identify isolates to a genuslevel.

Primer 1 Primer 2 Target V4_515F V4_806R The 4^(th) Variable region (5′-GTGCC (5′-GGACT of the bacterial 16S  AGCMGCCGC ACHVGGGTWrDNA (Caporaso, J.  GGTAA-3′) TCTAAT-3′) Gregory, et al. The  (SEQ ID(SEQ ID ISME journal 6.8 (2012): NO: 1450) NO: 1451) 1621-1624.) 27F1492R Full length of the (5′-AGAGT (5′-GGTTA bacterial 16S rDNA,TTGATCCTG CCTTGTTAC from position 8-1507. GCTCAG-3′) GACTT-3′) (SEQ ID(SEQ ID NO: 1452) NO: 1453) ITS1 ITS2 ~240 bp ITS1 region  (5′-TCCGT(5′-GCTGC of fungal genome AGGTGAACC GTTCTTCAT TGCGG-3′) CGATGC-3′)(SEQ ID (SEQ ID NO: 1454) NO: 1455) SR1R SR6 Small subunit (18s)(5′-TACCT (5′-TGTTA of the fungal rDNA GGTTGATTC CGACTTTTA geneTGCCAGT-3′) CTT-3′) (SEQ ID (SEQ ID NO: 1456) NO: 1457) ITS1F ITS4~600-1000 bp ITS  (5′-CTTGG (5′-TCCTC region of fungal  TCATTTAGACGCTTATTG genomes (J Microbiol GGAAGTAA-3′) ATATGC-3′)Methods. 2007 Oct; (SEQ ID (SEQ ID 71(1):7-14. Epub  NO: 1458) NO: 1459)2007 Jul 5.

To decrease background noise due to the non-specific binding of primersto DNA, the thermocycler is programmed for a touchdown-PCR, whichincreases specificity of the reaction at higher temperatures andincreases the efficiency towards the end by lowering the annealingtemperature. Below is an example of the conditions for a typicalTouchdown PCR.

Step # Cycle Temperature Time 1 Initial Denaturalization 98° C.*  5 m 2Denaturalization 98° C.* 30 s 3 Annealing Predicted optimal Tm for 30 sthe primer set +10° C., inus 1° C./cycle 4 Elongation 72° C.* 1 m/1 Kb 5GoTo Step 2 × 10 times 6 Denaturalization 98° C.* 30 s 7 AnnealingPredicted optimal Tm for 30 s the primer set 8 Elongation 72° C.* 1 m/1Kb 9 GoTo Step 6 × 20 times 10 Final Elongation 72° C.*  5 m 11 CoolDown 4° C.  5 m *Or the temperature specified by the DNA polymerasemanufacturer for this step.PCR reactions are purified to remove primers, dNTPs, and othercomponents by methods known in the art, for example by the use ofcommercially available PCR clean-up kits, or 3M sodium acetate andchilled absolute ethanol as described below:

-   -   1. For each 20 μL of PCR product, the following mixture is        prepared and maintained cool in a 1.5 mL Tube.        -   2 μL of 3M sodium acetate (NaOAc) pH 4.5        -   40 μL chilled absolute ethanol.    -   2. Transfer the PCR product (20 μl) into the tube containing the        mixture.    -   3. Vortex the tube and then store it at −20 for 30-50 min.    -   4. Spin for 30 min at maximum speed 14.000 rpm.    -   5. Remove the supernatant carefully without disturbing the        pellet. (Do not touch the bottom of the tube)    -   6. Wash the pellet with 100 μl of chilled 70% ethanol and        centrifuge at ˜13,000 RCF for 5 min.    -   7. Remove the supernatant and then dry the pellet using a vacuum        centrifuge or by leaving the tube open in a biosafety hood.    -   8. Resuspend the pellet in 30 μL in sterile water.

DNA amplicons are sequenced using methods known in the art, for exampleSanger sequencing (Johnston-Monje D, Raizada M N (2011) PLoS ONE 6(6):e20396) using one of the two primers used for amplification.

The resulting sequences are aligned as query sequences with the publiclyavailable databases GenBank nucleotide, RDP (Wang, Q, G. M. Garrity, J.M. Tiedje, and J. R. Cole. 2007. Appl Environ Microbiol.73(16):5261-7.), UNITE (Abarenkov, Nilsson et al. New Phytologist 2010;Volume 186: 281-285.) and PlutoF (Evol Bioinform Online. 2010; 6:189-196.). RDP is specifically compiled and used for bacterial 16sclassification. UNITE and PlutoF are specifically compiled and used foridentification of fungi. In all the cases, the strains are identified tospecies level if their sequences are more than 95% similar to anyidentified accession from all databases analyzed (Zimmerman, Naupaka B.,and Peter M. Vitousek. 109.32 (2012): 13022-13027.). When the similaritypercentage is between 90-97%, the strain is classified at genus, family,order, class, subdivision or phylum level depending on the informationdisplayed in databases used. Isolates with lower similarity values (from30-90%) are classified as unknown or uncultured depending on theinformation displayed after BLAST analysis. To support the molecularidentification, fungal taxa are confirmed by inducing sporulation on PDAor V8 agar plates and using reported morphological criteria foridentification of fruiting bodies structure and shape (Ainsworth, G.Geoffrey Clough. Ainsworth and Bisby's Dictionary of the Fungi. CABI,2008). Bacterial taxa are confirmed by using reported morphologicalcriteria in specialized differential media for the particular taxon, orby biochemical differentiation tests, as described by the Bergey'sManual of Systematic Microbiology (Whitman, William B., et al., eds.Bergey's Manual® of systematic bacteriology. Vols. 1-5. Springer, 2012).

Culture-Independent Characterization of Fungal and Bacterial Communitiesin Seeds or Plants

Example Description

To understand the diversity of culturable and unculturable microbial(bacterial and fungal) taxa that reside inside of seeds or plants ofagriculturally-relevant cultivars, landraces, and ancestral wildvarieties, microbial DNA is extracted from surface sterilized seed orplant parts, followed by amplification of conserved genomic regions likethe ribosomal DNA loci. Amplified DNA represents a “snapshot” of thefull microbial community inside seeds or plants.

Experimental Description

To obtain microbial DNA from seeds, plants or plant parts, the seeds,plants or plant parts are surface sterilized under aseptic conditions asdescribed herein. Microbial DNA from seeds, plants, or plant parts isextracted using methods known in the art, for example using commerciallyavailable Seed-DNA or plant DNA extraction kits, or the followingmethod.

-   -   1. A sample of each kind of seed or plant tissue is placed in a        cold-resistant container and 10-50 mL of liquid nitrogen is        applied. The seeds or plant tissues are then macerated to a        powder.    -   2. Genomic DNA is extracted from each seed or plant tissue        preparation, following a chloroform:isoamyl alcohol 24:1        protocol (Sambrook et al. 1989).

Fungal-specific primers are used to amplify the ITS (InternalTranscribed Spacer) region of nuclear ribosomal DNA (Schoch, Conrad L.,et al. Proceedings of the National Academy of Sciences 109.16 (2012):6241-6246.). Bacterial specific primers are used to amplify region ofthe 16s rDNA gene of the bacterial genome (Caporaso, J. Gregory, et al.The ISME journal 6.8 (2012): 1621-1624.). Sequences obtained through NGSplatforms are analyzed against databases, such as the ones mentionedherein.

Some of the primer pairs used for this analysis are detailed below:

Primer 1 Primer 2 Target V4_515F V4_806R The 4^(th) Variable (see above)(see above) region of the bacterial 16S rDNA 27F 1492RFull length of the (see above) (see above) bacterial 16S rDNA,from position 8-1507. ITS1 ITS2 ~240 bp ITS1 region (see above)(see above) of fungal genome SR1R SR6 Small subunit (18s) (see above)(see above) of the fungal rDNA gene ITS1F ITS4 ~600-1000 bp ITS(5′-CTTGG (5′-TCCTC region of fungal TCATTTAGA CGCTTATTGgenomes (J Microbiol GGAAGTAA-3′) ATATGC-3′)  Methods. 2007 Oct; (SEQ ID(SEQ ID 71(1):7-14. Epub NO: 1458) NO: 1459) 2007 Jul 5. ITS5 ITS4Asco~500 bp fragment (Universal) (Ascomycota- from different (5′-GGAAGTAspecific):  fungal Phyla AAAGTCGTAAC 5′ CGTTACTRR depending on primerAAGG-3′) GGCAATCCCTGT combination used. (SEQ ID TG 3′ Liliya G. NO: 1460) (SEQ ID  Nikolcheva, NO: 1461) or Felix Bärlocher ITS4BasidioMycological Progress (Basidiomycota- 01/2004; 3(1):41-49. specific):  5′GCRCGGAAR ACGCTTCTC 3′ (SEQ ID  NO: 1462); or ITS4Chytrid(Chytridiomycota- specific): 5′ TTTTCCCGT TTCATTCGCCA 3′ (SEQ IDNO: 1463); or ITS4Oo (Oomycota- specific): 5′ ATAGACTAC AATTCGCC 3′(SEQ ID NO: 1464); or ITS4Zygo (Zygomycota- specific): 5′ AAAACGTWTCTTCAAA 3′ (SEQ ID NO: 1465). SSUmAf- LSUmAr 1000-1600 bp fragment(equimolar (equimolar of the Glomerycota mix of 2 mix of 4 (arbusculardegenerate degenerate mycorrhizae) genome primers) and primers) andcomprising partial SSUmCf LSUmBr SSU, whole internal equimolar(equimolar transcribed spacer mix of 3 mix of 5 (ITS) rDNA region anddegenerate degenerate partial LSU. primers) primers) Manuela Kruger,Herbert, Claudia  Kruger and Arthur  SchtiBler. (2009) DNA-based specieslevel detection of Glomeromycota: one PCR primer set for all arbuscularmycorrhizal fungi. New Phytol. 183(1):212-23. Arch 340F Arch 1000R660 by product of (5′-CCCTA (5′-GAGARG the 18S from Archaea YGGGGYGCAWRGTGCATGG (Gantner, S., et al. SCAG-3′) CC-3′)  (2011). Journal of(SEQ ID (SEQ ID microbiological NO: 1466) NO: 1467) methods, 84(1),12-18.) 27F-Degen 27F-Degen Full length of the (5′-AGRRT (5′-HGGHTAbacterial 16S rDNA, TYGATYMTG CCTTGTTACG from position GYTYAG-3′)ACTT-3′) 8-1507. (SEQ ID (SEQ ID Johnston-Monje D, NO: 1468) NO: 1469)Raizada MN (2011) and 799f PLoS ONE 6(6): (5′-AACMG e20396. GATTAGATACCCKG-3′) (SEQ ID NO: 1470)

As an alternative to next generation sequencing, Terminal RestricitionFragment Length Polymorphism, (TRFLP) can be performed, essentially asdescribed in Johnston-Monje, D. and Raizada et al [PLoS ONE 6(6): e20396(2011)]. Group specific, fluorescently labeled primers are used toamplify diagnostic regions of genes in the microbial population. Thisfluorescently labeled PCR product is cut by a restriction enzyme chosenfor heterogeneous distribution in the PCR product population. The enzymecut mixture of fluorescently labeled and unlabeled DNA fragments is thensubmitted for sequence analysis on a Sanger sequence platform such asthe Applied Biosystems 3730 DNA Analyzer.

Determination of the Plant Pathogenic Potential of Microbial Isolates

Since a microbe which confers positive traits to one cultivar might be apathogenic agent in a different plant species, a general assay is usedto determine the pathogenic potential of the isolates. Surface andinterior-sterilized seeds are germinated in water agar, and once theplant develops its first set of leaves, are inoculated with the isolate.Alternatively, the plants are inoculated as seeds. For inoculation themicrobial isolate is grown on solid media, and inoculated into a plantor onto a seed via any of the methods described herein. Plants areallowed to grow under ideal conditions for 2-3 weeks and any pathogeniceffect of the introduced microbe is evaluated against uninoculatedcontrol plants.

Testing for Microbial Traits In Vitro

Examples below are adapted from: Johnston-Monje D, Raizada M N (2011)PLoS ONE 6(6): e20396, which is incorporated herein by reference in itsentirety.

Assay for Growth on Nitrogen Free LGI Media.

All glassware is cleaned with 6 M HCl before media preparation. A new 96deep-well plate (2 mL well volume) is filled with 1 mL/well of sterileLGI broth [per L, 50 g Sucrose, 0.01 g FeCl₃-6H₂O, 0.8 g K₃PO₄, 0.2 gMgSO₄-7H₂O, 0.002 g Na₂MoO₄-2H₂O, pH 7.5]. Bacteria are inoculated witha flame-sterilized 96 pin replicator. The plate is sealed with abreathable membrane, incubated at 25° C. with gentle shaking for 5 days,and OD₆₀₀ readings taken.

ACC Deaminase Activity Assay.

Microbes are assayed for growth with ACC as their sole source ofnitrogen. Prior to media preparation all glassware is cleaned with 6 MHCl. A 2 M filter sterilized solution of ACC (#1373A, Research Organics,USA) is prepared in water. 1 μl/mL of this is added to autoclaved LGIbroth (see above), and 1 mL aliquots are placed in a new 96 well plate.The plate is sealed with a breathable membrane, incubated at 25° C. withgentle shaking for 5 days, and OD600 readings taken. Only wells that aresignificantly more turbid than their corresponding nitrogen free LGIwells are considered to display ACC deaminase activity.

Mineral Phosphate Solubilization Assay.

Microbes are plated on tricalcium phosphate media. This is prepared asfollows: 10 g/L glucose, 0.373 g/L NH₄NO₃, 0.41 g/L MgSO₄, 0.295 g/LNaCl, 0.003 FeCl₃, 0.7 g/L Ca₃HPO₄ and 20 g/L Agar, pH 6, thenautoclaved and poured into 150 mm plates. After 3 days of growth at 25°C. in darkness, clear halos are measured around colonies able tosolubilize the tricalcium phosphate.

RNAse Activity Assay.

1.5 g of torula yeast RNA (#R6625, Sigma) is dissolved in 1 mL of 0.1 MNa₂HPO₄ at pH 8, filter sterilized and added to 250 mL of autoclaved R2Aagar media which is poured into 150 mm plates. The bacteria from aglycerol stock plate are inoculated using a flame-sterilized 96 pinreplicator, and incubated at 25° C. for 3 days. On day three, plates areflooded with 70% perchloric acid (#311421, Sigma) for 15 minutes andscored for clear halo production around colonies.

Acetoin and Diacetyl Production Assay.

1 mL of autoclaved R2A broth supplemented with 0.5% glucose is aliquotedinto a 96 deep well plate (#07-200-700, Fisher). The bacteria from aglycerol stock plate are inoculated using a flame-sterilized 96 pinreplicator, sealed with a breathable membrane, then incubated for 5 dayswith shaking (200 rpm) at 25° C. At day 5, 100 μl aliquots of cultureare removed and placed into a 96 well white fluorometer plate, alongwith 100 μl/well of Barritt's Reagents A and B which are prepared bymixing 5 g/L creatine mixed 3:1 (v/v) with freshly preparedalpha-naphthol (75 g/L in 2.5 M sodium hydroxide). After 15 minutes,plates are scored for red or pink colouration against a copper colourednegative control.

Auxin Production Assay.

R2A agar media, supplemented with L-tryptophan to a final concentrationof 5 mM, is autoclaved and poured into 150 mm plates. Using a 96 pinplate replicator, all microbes are inoculated onto the fresh plate froma 96 well plate glycerol stock. The plate is incubated at 25° C. for 3days, then overlaid with a nitrocellulose membrane, and put in a fridgeat 4° C. overnight, allowing bacteria and their metabolites toinfiltrate into the paper. The next day, the nitrocellulose membrane isremoved and placed for 30 min on Whatman #2 filter papers saturated withSalkowski reagent (0.01 M ferric chloride in 35% perchloric acid,#311421, Sigma). Dark pink halos around colonies are visualized in themembrane by background illumination using a light table.

Siderophore Production Assay.

To ensure no contaminating iron is carried over from previousexperiments, all glassware is deferrated with 6 M HCl and water prior tomedia preparation. In this cleaned glassware, R2A agar media, which isiron limited, is prepared and poured into 150 mm Petri dishes andinoculated with bacteria using a 96 pin plate replicator. After 3 daysof incubation at 25° C., plates are overlaid with 0-CAS overlay. Againusing the cleaned glassware, 1 liter of 0-CAS overlay is made by mixing60.5 mg of Chrome azurol S (CAS), 72.9 mg of hexadecyltrimethyl ammoniumbromide (HDTMA), 30.24 g of finely crushedPiperazine-1,4-bis-2-ethanesulfonic acid (PIPES) with 10 mL of 1 mMFeCl₃.6H₂O in 10 mM HCl solvent. The PIPES had to be finely powdered andmixed gently with stirring (not shaking) to avoid producing bubbles,until a dark blue colour is achieved. Melted 1% agarose is then added topre-warmed O-CAS just prior pouring the overlay in a proportion of 1:3(v/v). After 15 minutes, colour change is scored by looking for purplehalos (catechol type siderophores) or orange colonies (hydroxamatesiderophores).

Pectinase Activity Assay.

Adapting a previous protocol 0.2% (w/v) of citrus pectin (#76280, Sigma)and 0.1% triton X-100 are added to R2A media, autoclaved and poured into150 mm plates. Bacteria are inoculated using a 96 pin plate replicator.After 3 days of culturing in the darkness at 25° C., pectinase activityis visualized by flooding the plate with Gram's iodine. Positivecolonies are surrounded by clear halos.

Cellulase Activity Assay.

Adapting a previous protocol, 0.2% carboxymethylcellulose (CMC) sodiumsalt (#C5678, Sigma) and 0.1% triton X-100 are added to R2A media,autoclaved and poured into 150 mm plates. Bacteria are inoculated usinga 96 pin plate replicator. After 3 days of culturing in the darkness at25° C., cellulose activity is visualized by flooding the plate withGram's iodine. Positive colonies are surrounded by clear halos.

Antibiosis Assay.

Bacteria are inoculated using a 96 pin plate replicator onto 150 mmPetri dishes containing R2A agar, then grown for 3 days at 25° C. Atthis time, colonies of either E. coli DH5α (gram negative tester),Bacillus subtillus ssp. Subtilis (gram positive tester), or yeast strainAH109 (fungal tester) are resuspended in 1 mL of 50 mM Na₂HPO₄ buffer toan OD₆₀₀ of 0.2, and 30 μl of this is mixed with 30 mL of warm LB agar.This is quickly poured completely over a microbe array plate, allowed tosolidify and incubated at 37° C. for 16 hours. Antibiosis is scored bylooking for clear halos around microbial colonies.

Generating/Isolating Endophytes Compatible with Agrochemicals

The application of pesticides against fungal pathogens ofagriculturally-relevant plants is a common practice in agriculture toensure higher yields. One method of pesticide delivery is to cover theseeds with a coating with pesticides. Although pesticides are meant todeter the growth and propagation of pathogenic microorganisms, they mayalso affect endophyte populations residing inside of the seed. For thispurpose, conferring compatibility mechanisms to endophytic fungiproviding beneficial properties which are sensitive to these compoundsis desirable for the maintenance of endophytes in the seeds.

Compatibility with pesticides can be intrinsic (naturally pesticidecompatible fungi, for example) or acquired (due to mutations in thegenetic material of the microorganism, or to the introduction ofexogenous DNA by natural DNA transfer).

Fungicides used as protectants are effective only on the seed surface,providing protection against seed surface-borne pathogens and providingsome level of control of soil-borne pathogens. These products generallyhave a relatively short residual. Protectant fungicides such as captan,maneb, thiram, or fludioxonil help control many types of soil-bornepathogens, except root rotting organisms. Systemic fungicides areabsorbed into the emerging seedling and inhibit or kill susceptiblefungi inside host plant tissues. Systemic fungicides used for seedtreatment include the following: azoxystrobin, carboxin, mefenoxam,metalaxyl, thiabendazole, trifloxystrobin, and various triazolefungicides, including difenoconazole, ipconazole, tebuconazole, andtriticonazole. Mefenoxam and metalaxyl are primarily used to target theoomycetes such as species of Pythium and Phytophthora.

Strobilurin analogues, such as azoxystrobin, inhibit mitochondrialrespiration by blocking electron transfer at the cytochrome bc1 complex.Phenylamides, including metalaxyl, interfere with RNA synthesis intarget fungi. Oxathiin systemic fungicides like carboxin inhibits theincorporation of phenylalanine into protein and of uracil into RNA.Azole fungicides BAS 480F, flusilazole, and tebuconazole are inhibitorsof sterol 14α-demethylase, and block sterol biosynthesis.

Determination of Intrinsic Resilience Against Agrochemicals of BacteriaCultured from Seeds

To test the intrinsic resilience pesticides of bacteria isolated asdescribed herein, minimum inhibitory concentration (MIC) assays areperformed on all isolated bacteria of interest, as described in Wiegand,Irith, Kai Hilpert, and Robert E W Hancock. Nature protocols 3.2 (2008):163-175, which is incorporated herein by reference in its entirety.Briefly, known concentrations of bacterial cells or spores are used toinoculate plates containing solid media with different concentrations ofthe pesticide, or to inoculate liquid media containing differentconcentrations of the pesticide (in a 96-well plate). The pesticides areused at the concentration recommended by the manufacturer for seedcoating, and two-fold dilutions down to 0.000125 (12 two-folddilutions). Growth is assessed after incubation for a defined period oftime (16-20 h) and compared to cultures grown in the same manner withoutany pesticides as control. The MIC value is determined as described inWiegand, Irith, Kai Hilpert, and Robert E W Hancock. Nature protocols3.2 (2008): 163-175.

Determination of Intrinsic Resilience Against Agrochemicals of FungiCultured from Seeds

To test the intrinsic resilience against pesticides of the fungiisolated as described in this application, minimum inhibitoryconcentration (MIC) assays are performed on all isolated fungi ofinterest, as described in Mohiddin, F. A., and M. R. Khan. AfricanJournal of Agricultural Research 8.43 (2013): 5331-5334 (incorporatedherein by reference in its entirety), with the following changes:Briefly, double strength potato dextrose agar is prepared containingdifferent concentrations of each pesticide. The pesticides are appliedat the concentration recommended by the manufacturer, and also in twofold dilutions to 0.000125× (12 two-fold dilutions). Thereafter, theplates are seeded centrally with a 3 mm disc of 4 days old culture ofeach fungus that had been centrifuged and rinsed twice in sterilephosphate buffer. PDA plates without a fungicide but inoculated with thefungi serve as a control. The inoculated plates are incubated at 25±2°C. for 5 days. The radial growth of the colony in each treatment ismeasured and the percent inhibition of growth is calculated as describedby Mohiddin, F. A., and M. R. Khan. African Journal of AgriculturalResearch 8.43 (2013): 5331-5334 (incorporated herein by reference in itsentirety). Fungal isolates are classified as resilience against theparticular pesticide if their maximum tolerance concentration (MTC) is2× or above the concentration of pesticides recommended to be used inseed coatings.

Generating Fungal Species with Compatibility with Commercial PesticidesCoated onto Seeds

When a fungal strain of interest that provides a beneficial property toits plant host is found to be sensitive to a commercially-relevantpesticide, pesticide-compatible variants of the strains need to begenerated for use in this application. Generation of compatibility tomultiple pesticides or cocktails of pesticides is accomplished bysequentially selecting compatible variants to each individual pesticideand then confirming compatibility with a combination of the pesticides.After each round of selection, fungi are tested for their ability toform symbiotic relationships with the plants and to confirm that theyprovide a beneficial property on the plant as did the parental strain,with or without application of the pesticide product as describedherein.

Generation and isolation of pesticide-compatible strains derived fromendophytic strains isolated from seeds and shown to provide beneficiarytraits to plants is performed as described by Shapiro-Ilan, David I., etal. Journal of invertebrate pathology 81.2 (2002): 86-93 (incorporatedherein by reference in its entirety), with some changes. Briefly, sporesof the isolated fungi are collected and solutions containing between˜1×10³ spores are used to inoculate potato dextrose agar (PDA) platescontaining 2, 5, and 10 times the MTC of the particular strain. Platesare incubated for 1-5 days and a single colony from the highestconcentration of pesticide which allows growth is inoculated onto afresh plate with the same pesticide concentration 7 consecutive times.After compatibility has been established, the strain is inoculated ontoPDA plates 3 consecutive times and then inoculated onto a PDA platecontaining the pesticide to confirm that the compatibility trait ispermanent.

Alternatively, if this method fails to provide a compatible strain, aspore suspension is treated with ethyl methanesulfonate to generaterandom mutants, similarly as described by Leonard, Cory A., Stacy D.Brown, and J. Russell Hayman. International journal of microbiology 2013(2013), Article ID 901697 (incorporated herein by reference in itsentirety) and spores from this culture are used in the experimentdetailed above.

To develop fungal endophytes compatible with multiple pesticides orcocktails of pesticides, spores of a strain compatible with one or morepesticides are used to select for variants to a new pesticide asdescribed above. Strains developed this way are tested for retention ofthe pesticide-compatibility traits by inoculating these strains onto PDAplates containing each single pesticide or combinations of pesticides.

Generating Bacterial Species with Compatibility to Commercial PesticidesCoated onto Seeds

When a bacterial strain of interest is found to be sensitive to acommercially-relevant pesticide, generation of pesticide-compatiblevariants of the strains can be generated for use in this application.Generation of compatible with multiple pesticides or cocktails ofpesticides is accomplished by first sequentially selecting variantscompatible with incrementally higher concentrations of each individualpesticide (as described by Thomas, Louise, et al. Journal of HospitalInfection 46.4 (2000): 297-303, which is incorporated herein byreference in its entirety). To develop bacterial endophytes compatiblewith multiple pesticides or cocktails of pesticides, bacterial cells ofa strain compatible with one or more pesticides is used to select forvariants to a new pesticide as described above. Strains developed thisway are tested for retention of the pesticide-compatible traits byinoculating these strains onto PDA plates containing each singlepesticide or combinations of pesticides.

After each round of selection, bacteria are tested for their ability tolive within plants and for their ability to provide the same beneficialproperty to the plant as did the parental strain, with or withoutapplication of the pesticide product to the plants as described herein.

Generation of Pesticide-Compatible Bacteria by Insertion of a ResistancePlasmid

Many bacterial plasmids that confer compatible pesticides have beendescribed in the literature (Don, R. H., and J. M. Pemberton. Journal ofBacteriology 145.2 (1981): 681-686; and Fisher, P. R., J. Appleton, andJ. M. Pemberton. Journal of bacteriology 135.3 (1978): 798-804, each ofwhich is incorporated herein by reference in its entirety)

For cases in which obtaining naturally occurring compatible bacteria isnot feasible, use of these plasmids is a possible way to developendophytic strains compatible with multiple pesticides.

For example, a Pseudomonas fluorescens strain that providesanti-nematode properties to plants but is sensitive to the pesticides2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic can betransformed with the plasmid pJP2 (isolated from Alcaligenes eutrophus)which provides transmissible compatible with these compounds, asdescribed by Don and Pemberton, 1981. Briefly, plasmids are transferredby conjugation to Pseudomonas, using the method described in Haas,Dieter, and Bruce W. Holloway. Molecular and General Genetics 144.3(1976): 243-251 (incorporated herein by reference in its entirety).

After the generation of bacteria carrying pesticide-compatibilityconferring plasmids, these endophytes are tested for their ability tolive inside plant tissues and for their ability to provide the samebeneficial property to the plant as it did for the parental strain, withor without application of the pesticide product to the plants asdescribed herein.

Growth & Scale-Up of Bacteria for Inoculation on Solid Media

The bacterial isolates are grown by loop-inoculation of a single colonyinto R2A broth (supplemented with appropriate antibiotics) in 100 mLflasks. The bacterial culture is incubated at 30±2° C. for 2 days at 180rpm in a shaking incubator (or under varying temperatures and shakingspeeds as appropriate). This liquid suspension is then used to inoculateheat sterilized vermiculite powder which is premixed with sterile R2Abroth (without antibiotics), resulting in a soil like mixture ofparticles and liquid. This microbial powder is then incubated for anadditional couple of days at 30±2° C. with daily handshaking to aeratethe moist powder and allow bacterial growth. Microbially inoculatedvermiculite powder is now ready for spreading on to soil or onto plantparts. Alternatively, the R2A broth is used to inoculate Petri dishescontaining R2A or another appropriate nutrient agar where lawns ofbacteria are grown under standard conditions and the solid coloniesscraped off, resuspended in liquid and applied to plants as desired.

Growth & Scale-Up of Fungi for Inoculation on Solid Media

Once a fungal isolate has been characterized, conditions are optimizedfor growth in the lab and scaled-up to provide sufficient material forassays. For example, the medium used to isolate the fungus issupplemented with nutrients, vitamins, co-factors, plant-extracts, andother supplements that can decrease the time required to grow the fungalisolate or increase the yield of mycelia and/or spores the fungalisolate produces. These supplements can be found in the literature orthrough screening of different known media additives that promote thegrowth of all fungi or of the particular fungal taxa.

To scale up the growth of fungal isolates, isolates are grown from afrozen stock on several Petri dishes containing media that promotes thegrowth of the particular fungal isolate and the plates are incubatedunder optimal environmental conditions (temperature, atmosphere, light).After mycelia and spore development, the fungal growth is scraped andresuspended in 0.05M Phosphate buffer (pH 7.2, 10 mL/plate). Disposablepolystyrene Bioassay dishes (500 cm², Thermo Scientific NuncUX-01929-00) are prepared with 225 mL of autoclaved media with anyrequired supplements added to the media, and allowed to solidify. Platesare stored at room temperature for 2-5 days prior to inoculation toconfirm sterility. 5 mL of the fungal suspension is spread over thesurface of the agar in the Bioassay plate in a biosafety cabinet, platesare allowed to dry for 1 h, and they are then incubated for 2-5 days, oruntil mycelia and/or spores have developed.

A liquid fungal suspension is then created via the following. Fungalgrowth on the surface of the agar in the Bioassay plates are thenscraped and resuspended in 0.05M Phosphate buffer (pH 7.2). OD₆₀₀readings are taken using a spectrometer and correlated to previouslyestablished OD₆₀₀/CFU counts to estimate fungal population densities,and the volume adjusted with additional sodium phosphate buffer toresult in 100 mL aliquots of fungi at a density of approximately10⁶-10¹¹ spores/mL. This suspension may or may not be filtered to removemycelia and can be used to create a liquid microbial formulation asdescribed herein to apply the fungal isolate onto a plant, plant part,or seed.

Growth & Scale-Up of Bacteria for Inoculation in Liquid Media

Bacterial strains are grown by loop-inoculation of one single colonyinto R2A broth (amended with the appropriate antibiotics) in 100 Lflasks. The bacterial culture is incubated at 30±2° C. for 2 days at 180rpm in a shaking incubator (or under varying temperatures and shakingspeeds as appropriate). The bacteria are pelleted by centrifugation andresuspended in sterile 0.1 M sodium phosphate. OD₆₀₀ readings are takenusing a spectrometer and correlated to previously established OD₆₀₀/CFUcounts to estimate bacterial population densities, and the volumeadjusted with additional sodium phosphate buffer to result in 100 mLaliquots of bacteria at a density of 1×10⁸ cells/mL. To help breaksurface tension, aid bacterial entry into plants and provide microbesfor some energy for growth, 10 μL of Silwet L-77 surfactant and 1 g ofsucrose is added to each 100 mL aliquot (resulting in 0.01% v/v and 1%v/v concentrations, respectively) in a similar way as in the protocolfor Agrobacterium-mediated genetic transformation of Arabidopsisthaliana seed [Clough, S., Bent, A. (1999) The Plant Journal 16(4735-743].

Growth & Scale-Up of Fungi for Inoculation in Liquid Media

Once a fungal isolate has been characterized, conditions are optimizedfor growth in the lab and scaled-up to provide enough material forassays. For example, the medium used to isolate the fungi issupplemented with nutrients, vitamins, co-factors, plant-extracts,and/or other supplements that can decrease the time required to grow thefungal isolate and/or increase the yield of mycelia and/or spores thefungal isolate produces. These supplements can be found in theliterature or through screening of different known media additives thatpromote the growth of all fungi or of the particular fungal taxa.

To scale up the growth of fungal isolates, isolates are grown from afrozen stock on Petri dishes containing media that promotes the growthof the particular fungal isolate and the plates are incubated underoptimal environmental conditions (temperature, atmosphere, light). Aftermycelia and spore development, the fungal growth is scraped andresuspended in 0.05M Phosphate buffer (pH 7.2, 10 mL/plate). 1 liter ofliquid media selected to grow the fungal culture is prepared in 2 Lglass flasks and autoclaved and any required supplements added to themedia. These are stored at room temperature for 2-5 days prior toinoculation to confirm sterility. 1 mL of the fungal suspension is addedaseptically to the media flask, which is then incubated for 2-5 days, oruntil growth in the liquid media has reached saturation. OD₆₀₀ readingsare taken using a spectrometer and correlated to previously establishedOD₆₀₀/CFU counts to estimate fungal population densities, and the volumeadjusted with additional sodium phosphate buffer to result in 100 mLaliquots of fungi at a density of approximately 10⁶-10¹¹ spores/mL. Thissuspension may or may not be filtered to remove mycelia and can be usedto create a liquid microbial formulation as described herein to applythe fungal isolate onto a plant, plant part, or seed.

Creation of Liquid Microbial Formulations or Preparations for theApplication of Microbes to Plants

Bacterial or fungal cells are cultured in liquid nutrient broth mediumto between 10²-10¹² CFU/mL. The cells are separated from the medium andsuspended in another liquid medium if desired. The microbial formulationmay contain one or more bacterial or fungal strains. The resultingformulation contains living cells, lyophilized cells, or spores of thebacterial or fungal strains. The formulation may also contain water,nutrients, polymers and binding agents, surfactants or polysaccharidessuch as gums, carboxymethylcellulose and polyalcohol derivatives.Suitable carriers and adjuvants can be solid or liquid and includenatural or regenerated mineral substances, solvents, dispersants,wetting agents, tackifiers, thickeners, binders or fertilizers.Compositions can take the form of aqueous solutions, oil-in-wateremulsions, or water-in-oil emulsions. Small amounts of insolublematerial can optionally be present, for example in suspension in themedium, but it is generally preferred to minimize the presence of suchinsoluble material.

Inoculation of Plants by Coating Microbes Directly onto Seed

Seed is treated by coating it with a liquid microbial formulation(prepared as described herein) comprising microbial cells and otherformulation components, directly onto the seed surface at the rate of10²-10⁸ microbial CFU per seed. Seeds are soaked in liquid microbialformulation for 1, 2, 3, 5, 10, 12, 18 or 24 hours or 2, 3, or 5 days.After soaking in microbial formulation, seeds are planted in growingcontainers or in an outdoor field. Seeds may also be coated with liquidmicrobial formulation by using an auger or a commercial batch treater.One or more microbial formulations or other seed treatments are appliedconcurrently or in sequence. Treatment is applied to the seeds using avariety of conventional treatment techniques and machines, such asfluidized bed techniques, augers, the roller mill method, rotostaticseed treaters, and drum coaters. Other methods, such as spouted beds mayalso be useful. The seeds are pre-sized before coating. Optionally themicrobial formulation is combined with an amount of insecticide,herbicide, fungicide, bactericide, or plant growth regulator, or plantmicro- or macro-nutrient prior to or during the coating process. Aftercoating, the seeds are typically dried and then transferred to a sizingmachine for grading before planting. Following inoculation, colonizationof the plants or seeds produced therefrom is confirmed via any of thevarious methods described herein. Growth promotion or stress resiliencebenefits to the plant are tested via any of the plant growth testingmethods described herein.

Inoculation of Plants with a Combination of Two or More Microbes

Seeds can be coated with bacterial or fungal endophytes. This methoddescribes the coating of seeds with two or more bacterial or fungalstrains. The concept presented here involves simultaneous seed coatingof two microbes (e.g., both a gram negative endophytic bacteriumBurkholderia phytofirmans and a gram positive endophytic bacteriumBacillus mojavensis). Optionally, both microbes are geneticallytransformed by stable chromosomal integration as follows. Bacillusmojavensis are transformed with a construct with a constitutive promoterdriving expression of a synthetic operon of GFPuv and spectinomycinresistance genes, while Burkholderia phytofirmans are transformed with aconstruct with a constitutive promoter driving expression of the lacoperon with an appended spectinomycin resistance gene. Seeds are coatedwith a prepared liquid formulation of the two microbes the variousmethods described herein. Various concentrations of each endophyte inthe formulation is applied, from 10²/seed to about 10⁸/seed. Followinginoculation, colonization of the plants or seeds produced therefrom maybe confirmed via any of the various methods described herein. Growthpromotion or stress resilience benefits to the plant are tested via anyof the plant growth testing methods described herein.

Culturing to Confirm Colonization of Plant by Bacteria

The presence in the seeds or plants of GFPuv or gusA-labeled endophytescan be detected by colony counts from mashed seed material andgerminated seedling tissue using R2A plates amended with5-Bromo-4-chloro-3-indolyl β-D-glucuronide (X-glcA, 50 μg/mL), IPTG (50μg/mL) and the antibiotic spectinomycin (100 μg/mL). Alternatively,bacterial or fungal endophytes not having received transgenes can alsobe detected by isolating microbes from plant, plant tissue or seedhomogenates (optionally surface-sterilized) on antibiotic free media andidentified visually by colony morphotype and molecular methods describedherein. Representative colony morphotypes are also used in colony PCRand sequencing for isolate identification via ribosomal gene sequenceanalysis as described herein. These trials are repeated twice perexperiment, with 5 biological samples per treatment.

Culture-Independent Methods to Confirm Colonization of the Plant orSeeds by Bacteria or Fungi

Example Description

One way to detect the presence of endophytes on or within plants orseeds is to use quantitative PCR (qPCR). Internal colonization by theendophyte can be demonstrated by using surface-sterilized plant tissue(including seed) to extract total DNA, and isolate-specific fluorescentMGB probes and amplification primers are used in a qPCR reaction. Anincrease in the product targeted by the reporter probe at each PCR cycletherefore causes a proportional increase in fluorescence due to thebreakdown of the probe and release of the reporter. Fluorescence ismeasured by a quantitative PCR instrument and compared to a standardcurve to estimate the number of fungal or bacterial cells within theplant.

Experimental Description

The design of both species-specific amplification primers, andisolate-specific fluorescent probes are well known in the art. Planttissues (seeds, stems, leaves, flowers, etc.) are pre-rinsed and surfacesterilized using the methods described herein.

Total DNA is extracted using methods known in the art, for example usingcommercially available Plant-DNA extraction kits, or the followingmethod.

-   -   1. Tissue is placed in a cold-resistant container and 10-50 mL        of liquid nitrogen is applied. Tissues are then macerated to a        powder.    -   2. Genomic DNA is extracted from each tissue preparation,        following a chloroform:isoamyl alcohol 24:1 protocol (Sambrook,        Joseph, Edward F. Fritsch, and Thomas Maniatis. Molecular        cloning. Vol. 2. New York: Cold spring harbor laboratory press,        1989.).

Quantitative PCR is performed essentially as described by Gao, Zhan, etal. Journal of clinical microbiology 48.10 (2010): 3575-3581 withprimers and probe(s) specific to the desired isolate using aquantitative PCR instrument, and a standard curve is constructed byusing serial dilutions of cloned PCR products corresponding to thespecie-specific PCR amplicon produced by the amplification primers. Dataare analyzed using instructions from the quantitative PCR instrument'smanufacturer software.

As an alternative to qPCR, Terminal Restriction Fragment LengthPolymorphism, (TRFLP) can be performed, essentially as described inJohnston-Monje D, Raizada M N (2011) PLoS ONE 6(6): e20396. Groupspecific, fluorescently labelled primers are used to amplify a subset ofthe microbial population, especially bacteria, especially fungi,especially archaea, especially viruses. This fluorescently labelled PCRproduct is cut by a restriction enzyme chosen for heterogeneousdistribution in the PCR product population. The enzyme cut mixture offluorescently labelled and unlabeled DNA fragments is then submitted forsequence analysis on a Sanger sequence platform such as the AppliedBiosystems 3730 DNA Analyzer.

Immunological Methods to Detect Microbes in Seeds and Vegetative Tissues

A polyclonal antibody is raised against specific bacteria X or fungus Ystrains via standard methods. A polyclonal antibody is also raisedagainst specific GUS and GFP proteins via standard methods.Enzyme-linked immunosorbent assay (ELISA) and immunogold labeling isalso conducted via standard methods, briefly outlined below.

Immunofluorescence microscopy procedures involve the use of semi-thinsections of seed or seedling or adult plant tissues transferred to glassobjective slides and incubated with blocking buffer (20 mMTris(hydroxymethyl)-aminomethane hydrochloride (TBS) plus 2% bovineserum albumin, pH 7.4) for 30 min at room temperature. Sections arefirst coated for 30 min with a solution of primary antibodies and thenwith a solution of secondary antibodies (goat anti-rabbit antibodies)coupled with fluorescein isothiocyanate (FITC) for 30 min at roomtemperature. Samples are then kept in the dark to eliminate breakdown ofthe light-sensitive FITC. After two 5-min washings with sterilepotassium phosphate buffer (PB) (pH 7.0) and one with double-distilledwater, sections are sealed with mounting buffer (100 mL 0.1 M sodiumphosphate buffer (pH 7.6) plus 50 mL double-distilled glycerine) andobserved under a light microscope equipped with ultraviolet light and aFITC Texas-red filter.

Ultrathin (50- to 70-nm) sections for TEM microscopy are collected onpioloform-coated nickel grids and are labeled with 15-nm gold-labeledgoat anti-rabbit antibody. After being washed, the slides are incubatedfor 1 h in a 1:50 dilution of 5-nm gold-labeled goat anti-rabbitantibody in IGL buffer. The gold labeling is then visualized for lightmicroscopy using a BioCell silver enhancement kit. Toluidine blue(0.01%) is used to lightly counterstain the gold-labeled sections. Inparallel with the sections used for immunogold silver enhancement,serial sections are collected on uncoated slides and stained with 1%toluidine blue. The sections for light microscopy are viewed under anoptical microscope, and the ultrathin sections are viewed by TEM.

Characterization of Uniformity of Endophytes in a Population of Seeds

To test for the homogeneity of endophytes either on the surface orcolonizing the interior tissues in a population of seeds, seeds aretested for the presence of the microbes by culture-dependent and/or-independent methods. Seeds are obtained, surface sterilized andpulverized, and the seed homogenate is divided and used to inoculateculture media or to extract DNA and perform quantitative PCR. Thehomogeneity of colonization in a population of seeds is assessed throughdetection of specific microbial strains via these methods and comparisonof the culture-dependent and culture-independent results across thepopulation of seeds. Homogeneity of colonization for a strain ofinterest is rated based on the total number of seeds in a populationthat contain a detectable level of the strain, on the uniformity acrossthe population of the number of cells or CFU of the strain present inthe seed, or based on the absence or presence of other microbial strainsin the seed.

Experimental Description

Surface sterilized seeds are obtained as described herein. Forculture-dependent methods of microbial-presence confirmation, bacterialand fungi are obtained from seeds essentially as described herein withthe following modification. Seed homogenate is used to inoculate mediacontaining selective and/or differential additives that will allow toidentification of a particular microbe.

For qPCR, total DNA of each seed is extracted using methods known in theart, as described herein.

Characterization of Homogeneity of Colonization in Population of Plants

To test for the homogeneity of microorganisms (including endophytes)colonizing the interior in a population of plants, tissues from eachplant are tested for the presence of the microbes by culture-dependentand/or -independent methods. Tissues are obtained, surface sterilizedand pulverized, and the tissue material is divided and used to inoculateculture media or to extract DNA and perform quantitative PCR. Thehomogeneity of colonization in a population of plants is assessedthrough detection of specific microbial strains via these methods andcomparison of the culture-dependent and culture-independent resultsacross the population of plants or their tissues. Homogeneity ofcolonization for a strain of interest is rated based on the total numberof plants in a population that contain a detectable level of the strain,on the uniformity across the population of the number of cells or CFU ofthe strain present in the plant tissue, or based on the absence orpresence of other microbial strains in the plant.

Experimental Description

Surface sterilized plant tissues are obtained as described herein. Forculture-dependent methods of microbial-presence confirmation, bacterialand fungi are obtained from plant tissues essentially as describedherein with the following modification. Plant tissue homogenate is usedto inoculate media containing selective and/or differential additivesthat will allow identification of a particular microbe.

For qPCR, total DNA of each plant tissue is extracted using methodsknown in the art, as described herein.

Testing for Beneficial and Inhibitory Effects of Endophytes

Testing for Germination Enhancement in Normal Conditions

Standard Germination Tests are used to assess the ability of theendophyte to enhance the seeds' germination and early growth. Briefly,400 seeds which are coated with the endophyte as described elsewhere areplaced in between wet brown paper towels (8 replicates with 50 seedseach). An equal number of seeds obtained from control plants that do notcontain the microbe is treated in the same way. The paper towels areplaced on top of 1×2 feet plastic trays and maintained in a growthchamber set at 25° C. and 70% humidity for 7 days. The proportion ofseeds that germinate successfully is compared between the seeds comingfrom microbe-colonized plants with those coming from controls.

Testing for Germination Enhancement Under Biotic Stress

A modification of the method developed by Hodgson [Am. Potato. J. 38:259-264 (1961)] is used to test germination enhancement inmicrobe-colonized seeds under biotic stress. Biotic stress is understoodas a concentration of inocula in the form of cell (bacteria) or sporesuspensions (fungus) of a known pathogen for a particular crop (e.g.,Pantoea stewartii or Fusarium graminearum for Zea mays L.). Briefly, foreach level of biotic stress, 400 seeds, the interiors of which arecolonized by microbial strains, and 400 seed controls (lacking themicrobial strains), are placed in between brown paper towels: 8replicates with 50 seeds each for each treatment (microbe-colonized andcontrol). Each one of the replicates is placed inside a large petri dish(150 mm in diameter). The towels are then soaked with 10 mL of pathogencell or spore suspension at a concentration of 10⁴ to 10⁸ cells/sporesper mL. Each level corresponds with an order of magnitude increment inconcentration (thus, 5 levels). The petri dishes are maintained in agrowth chamber set at 25° C. and 70% humidity for 7 days. The proportionof seeds that germinate successfully is compared between the seedscoming from microbe-colonized plants with those coming from controls foreach level of biotic stress.

Testing for Germination Enhancement Under Drought Stress

Polyethylene glycol (PEG) is an inert, water-binding polymer with anon-ionic and virtually impermeable long chain [Couper and Eley, J.Polymer Sci., 3: 345-349 (1984)] that accurately mimics drought stressunder dry-soil conditions. The higher the concentration of PEG, thelower the water potential achieved, thus inducing higher water stress ina watery medium. To determine germination enhancement in seeds, theinteriors of which are colonized by microbial strains, the effect ofosmotic potential on germination is tested at a range of water potentialrepresentative of drought conditions following Perez-Fernandez et al.[J. Environ. Biol. 27: 669-685 (2006)]. The range of water potentialssimulates those that are known to cause drought stress in a range ofcultivars and wild plants, (−0.05 MPa to −5 MPa) [Craine et al., NatureClimate Change 3: 63-67 (2013)]. The appropriate concentration ofpolyethylene glycol (6000) required to achieve a particular waterpotential is determined following Michel and Kaufmann (Plant Physiol.,51: 914-916 (1973)) and further modifications by Hardegree and Emmerich(Plant Physiol., 92, 462-466 (1990)). The final equation used todetermine amounts of PEG is: Ψ=0.130 [PEG]² T−13.7 [PEG]²; where theosmotic potential (Ψ) is a function of temperature (T). Germinationexperiments are conducted in 90 mm diameter petri dishes. Replicatesconsist of a Petri dish, watered with 10 mL of the appropriate solutionand 20 seeds floating in the solution. 400 seeds, the interiors of whichare colonized by microbial strains are tested, in addition to 400 seedcontrols (lacking the microbial strains), totaling 40 petri dishes. Toprevent large variations in Ψ, dishes are sealed with parafilm and thePEG solutions are renewed weekly by pouring out the existing PEG in thepetri dish and adding the same amount of fresh solution. Petri dishesare maintained in a growth chamber set at 25° C., 16:8 hour light:darkcycle, 70% humidity, and least 120 μE/m²/s light intensity. Theproportion of seeds that germinate successfully after two weeks iscompared between the seeds coming from inoculated plants and thosecoming from controls.

Testing for Germination Enhancement in Heat Conditions

Standard Germination Tests are used to determine if a microbe colonizingthe interior of a seed protects maize against heat stress duringgermination. Briefly, 400 seeds, the interiors of which are colonized bymicrobial strains are placed in between wet brown paper towels (8replicates with 50 seeds each). An equal number of seeds obtained fromcontrol plants that lack the microbe is treated in the same way. Thepaper towels are placed on top of 1×2 ft plastic trays and maintained ina growth chamber set at 16:8 hour light:dark cycle, 70% humidity, and atleast 120 μE/m2/s light intensity for 7 days. A range of hightemperatures (from 35° C. to 45° C., with increments of 2 degrees perassay) is tested to assess the germination of microbe-colonized seeds ateach temperature. The proportion of seeds that germinate successfully iscompared between the seeds coming from microbe-colonized plants andthose coming from controls.

Testing for Germination Enhancement in Cold Conditions

Standard Germination Tests are used to determine if a microbe colonizingthe interior of a seed protects maize against cold stress duringgermination. Briefly, 400 seeds, the interiors of which are colonized bymicrobial strains are placed in between wet brown paper towels (8replicates with 50 seeds each). An equal number of seeds obtained fromcontrol plants that lack the microbe is treated in the same way. Thepaper towels are placed on top of 1×2 ft plastic trays and maintained ina growth chamber set at 16:8 hour light:dark cycle, 70% humidity, and atleast 120 μE/m2/s light intensity for 7 days. A range of lowtemperatures (from 0° C. to 10° C., with increments of 2 degrees perassay) is tested to assess the germination of microbe-colonized seeds ateach temperature. The proportion of seeds that germinate successfully iscompared between the seeds coming from microbe-colonized plants andthose coming from controls.

Testing for Germination Enhancement in High Salt Concentrations

Germination experiments are conducted in 90 mm diameter petri dishes.Replicates consist of a Petri dish, watered with 10 mL of theappropriate solution and 20 seeds floating in the solution. 400 seeds,the interiors of which are colonized by microbial strains, and 400 seedcontrols (lacking the microbial strains) are tested in this way (40petri dishes total). To prevent large variations in salt concentrationdue to evaporation, dishes are sealed with parafilm and the salinesolutions are renewed weekly by pouring out the existing saline solutionin the petri dish and adding the same amount of fresh solution. A rangeof saline solutions (100-500 mM NaCl) is tested for to assess thegermination of microbe-colonized seeds at varying salt levels. Petridishes are maintained in a growth chamber set at 25° C., 16:8 hourlight:dark cycle, 70% humidity, and at least 120 μE/m2/s lightintensity. The proportion of seeds that germinates successfully aftertwo weeks is compared between the seeds coming from inoculated plantsand those coming from controls.

Testing for Germination Enhancement in Soils with High Metal Content

Standard Germination Tests are used to determine if a microbe colonizingthe interior of a seed protects maize against stress due to high soilmetal content during germination. Briefly, 400 seeds of maize, theinteriors of which are colonized by microbial strains, are placed inbetween wet brown paper towels (8 replicates with 50 seeds each). Anequal number of seeds obtained from control plants that lack the microbe(microbe-free) is treated in the same way. The paper towels are placedon top of 1×2 ft plastic trays with holes to allow water drainage. Thepaper towels are covered with an inch of sterile sand. For each metal tobe tested, the sand needs to be treated appropriately to ensure therelease and bioavailability of the metal. For example, in the case ofaluminum, the sand is watered with pH 4.0+˜1 g/Kg soil Al+3 (−621 uM).The trays are maintained in a growth chamber set at 25° C. and 70%humidity for 7 days. The proportion of seeds that germinatessuccessfully is compared between the seeds coming from microbe-colonizedplants and those coming from controls.

Testing for Growth Promotion in Growth Chamber in Normal Conditions

Soil is made from a mixture of 60% Sunshine Mix #5 (Sun Gro; Bellevue,Wash., USA) and 40% vermiculite. To determine if a particular microbecolonizing the interior of seeds is capable of promoting plant growthunder normal conditions, 24 pots are prepared in two 12-pot no-hole flattrays with 28 grams of dry soil in each pot, and 2 L of filtered wateris added to each tray. The water is allowed to soak into the soil andthe soil surface is misted before seeding. For each seed-microbecombination, 12 pots are seeded with 3-5 seeds colonized by the microbeand 12 pots are seeded with 3-5 seeds lacking the microbe (microbe-freeplants). The seeded pots are covered with a humidity dome and kept inthe dark for 3 days, after which the pots are transferred to a growthchamber set at 25° C., 16:8 hour light:dark cycle, 70% humidity, and atleast 120 μE/m2/s light intensity. The humidity domes are removed on day5, or when cotyledons are fully expanded. After removal of the domes,each pot is irrigated to saturation with 0.5× Hoagland's solution, thenallowing the excess solution to drain. Seedlings are then thinned to 1per pot. In the following days, the pots are irrigated to saturationwith filtered water, allowing the excess water to drain after about 30minutes of soaking, and the weight of each 12-pot flat tray is recordedweekly. Canopy area is measured at weekly intervals. Terminal plantheight, average leaf area and average leaf length are measured at theend of the flowering stage. The plants are allowed to dry and seedweight is measured. Significance of difference in growth betweenmicrobe-colonized plants and controls lacking the microbe is assessedwith the appropriate statistical test depending on the distribution ofthe data at p<0.05.

Testing for Growth Promotion in Growth Chamber Under Biotic Stress

Soil is made from a mixture of 60% Sunshine Mix #5 (Sun Gro; Bellevue,Wash., USA) and 40% vermiculite. To determine if a particular microbecolonizing the interior of seeds is capable of promoting plant growth inthe presence of biotic stress, 24 pots are prepared in two 12-potno-hole flat trays with 28 grams of dry soil in each pot, and 2 L offiltered water is added to each tray. The water is allowed to soak intothe soil before planting. For each seed-microbe combination test, 12pots are seeded with 3-5 seeds colonized by the microbe and 12 pots areseeded with 3-5 seeds lacking the microbe (microbe-free plants). Theseeded pots are covered with a humidity dome and kept in the dark for 3days, after which the pots are transferred to a growth chamber set at25° C., 16:8 hour light:dark cycle, 70% humidity, and at least 120μE/m2/s light intensity. The humidity domes are removed on day 5, orwhen cotyledons are fully expanded. After removal of the domes, each potis irrigated to saturation with 0.5× Hoagland's solution, allowing theexcess solution to drain. Seedlings are then thinned to 1 per pot. Inthe following days, the pots are irrigated to saturation with filteredwater, allowing the excess water to drain after about 30 minutes ofsoaking.

Several methods of inoculation are used depending on the lifestyle ofthe pathogen. For leaf pathogens (e.g., Pseudomonas syringeae orColletotrichum graminicola), a suspension of cells for bacteria (10⁸cell/mL) or spores for fungi (10⁷ spores/mL) is applied with anapplicator on the adaxial surface of each of the youngest fully expandedleaves. Alternatively for fungal pathogens that do not form conidiaeasily, two agar plugs containing mycelium (˜4 mm in diameter) areattached to the adaxial surface of each of the youngest leaves on eachside of the central vein. For vascular pathogens (e.g., Pantoeastewartii or Fusarium moniliforme), the suspension of cells or spores isdirectly introduced into the vasculature (5-10 μL) through a minorinjury inflected with a sterile blade. Alternatively, the seedlings canbe grown hydroponically in the cell/spore or mycelium suspension. Totest the resilience of the plant-microbe combination against insectstresses, such as thrips or aphids, plants are transferred to aspecially-designated growth chamber containing the insects. Soil-borneinsect or nematode pathogens are mixed into or applied topically to thepotting soil. In all cases, care is taken to contain the fungal, insect,nematode or other pathogen and prevent release outside of the immediatetesting area.

The weight of each 12-pot flat tray is recorded weekly. Canopy area ismeasured at weekly intervals. Terminal plant height, average leaf areaand average leaf length are measured at the cease of flowering. Theplants are allowed to dry and seed weight is measured. Significance ofdifference in growth between microbe-colonized plants and controlslacking the microbe is assessed with the appropriate statistical testdepending on the distribution of the data at p<0.05.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A synthetic combination comprising a purifiedbacterial population in association with a seed of a cereal agriculturalplant, wherein the purified bacterial population comprises a seedbacterial endophyte that is heterologous to the seed, has a 16S nucleicacid sequence that is at least 97% identical to a 16S nucleic acidsequence selected from the group consisting of SEQ ID NO: 521, 533, 560,588, 658, 710, 856 and 1447, and is capable of producing an auxin oracetoin, and wherein the seed bacterial endophyte is present in thesynthetic combination at a concentration of at least 10³ CFU/seed on thesurface of a seed and in an amount effective to increase tolerance todrought of a cereal agricultural plant produced from the seed ascompared to a reference plant grown under the same conditions.
 2. Apopulation comprising at least 1000 synthetic combinations of claim 1,wherein the synthetic combinations are disposed within a package and areshelf stable.
 3. A population comprising at least 1000 cerealagricultural plants produced by planting a population of syntheticcombinations of claim 1, wherein the plants are grown in a field.
 4. Thesynthetic combination of claim 1, wherein the seed bacterial endophyteis obtainable from an interior seed compartment.
 5. The syntheticcombination of claim 1, wherein the seed bacterial endophyte isobtainable from an exterior surface of a seed.
 6. The syntheticcombination of claim 1, wherein the seed bacterial endophyte isobtainable from a different cultivar, variety or crop as compared to theseed.
 7. The synthetic combination of claim 1, wherein the seedbacterial endophyte colonizes the roots of the cereal agriculturalplant.
 8. The synthetic combination of claim 1, wherein the seedbacterial endophyte is obtained from rice seed, maize seed, wheat seed,or barley seed.
 9. The synthetic combination of claim 1, wherein thesynthetic combination further comprises a purified fungal population.10. The synthetic combination of claim 1, wherein the bacterialpopulation includes two or more bacterial endophytes.
 11. A syntheticcombination comprising a purified bacterial population in associationwith a seed of a cereal agricultural plant, wherein the purifiedbacterial population comprises a first bacterial endophyte and a secondbacterial endophyte, wherein the first bacterial endophyte and secondbacterial endophyte has a 16S nucleic acid sequence that is at least 97%identical to a 16S nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 521, 533, 560, 588, 658, 710, 856 and 1447, andis capable of producing an auxin or acetoin, wherein the first bacterialendophyte and the second bacterial endophyte have different 16S nucleicacid sequence, and wherein the first bacterial endophyte and secondbacterial endophyte are present in the synthetic combination at aconcentration of at least 10³ CFU/seed on the surface of a seed and inan amount effective to increase tolerance to drought of a cerealagricultural plant produced from the seed as compared to a referenceplant grown under the same conditions.
 12. A population comprising atleast 1000 synthetic combinations of claim 11, wherein the syntheticcombinations are disposed within a package and are shelf stable.
 13. Apopulation comprising at least 1000 cereal agricultural plants producedby planting a population of synthetic combinations of claim 11, whereinthe plants are grown in a field.
 14. The synthetic combination of claim11, wherein the seed bacterial endophyte is obtainable from an interiorseed compartment.
 15. The synthetic combination of claim 11, wherein theseed bacterial endophyte is obtainable from an exterior surface of aseed.
 16. The synthetic combination of claim 11, wherein the seedbacterial endophyte is obtainable from a different cultivar, variety orcrop as compared to the seed.
 17. The synthetic combination of claim 11,wherein the seed bacterial endophyte colonizes the roots of the cerealagricultural plant.
 18. The synthetic combination of claim 11, whereinthe seed bacterial endophyte is obtained from rice seed, maize seed,wheat seed, or barley seed.
 19. The synthetic combination of claim 11,wherein the synthetic combination further comprises a purified fungalpopulation.
 20. The synthetic combination of claim 11, wherein thebacterial population comprises more than two bacterial endophytes.